Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl− absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle’s syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle’s variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle’s mutation increased total Cl− absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl− absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.The intercalated cell Cl For antigen retrieval, sections were boiled in 0.01 M Citrate Buffer (pH 6.0) for 10 min at 98 °C and then stored in ice-cold PBS for subsequent immunostaining. Consecutive, 3–4 mm cryosections were prepared from archived frozen mouse and rat kidneys . Immunoblot band density was normalized to Coomassie gel band density in the region similar mobility. To confirm protein loading, actin immunoreactivity was quantified in the same blots using a rabbit, anti-actin antibody .Lysate proteins were separated by SDS-PAGE on 8.5% acrylamide gels and then electroblotted to PVDF membranes . Blots were blocked with Odyssey Blocking Buffer (LI-COR Biosciences) following the manufacturer’s instructions and then incubated with primary (pendrin) antibody  overnight-test was used. To compare multiple groups, one-way ANOVA was used with a Holm-Sidak post-test, as appropriate (see figure legends). The criterion for statistical significance was P < 0.05.Data are presented as the mean ± SE. Each “n” used in the statistical analysis represents data from separate animals. Statistical tests were performed with SigmaPlot 12.5 software. To test for statistical significance between two groups, an unpaired Student’s Scnn1a transcript has been detected in ICs [Scnn1aloxlox) and collecting duct-specific αENaC null mice  [+-ATPase or for αENaC and pendrin. In both the floxed αENaC  and in the collecting duct-specific αENaC null mice , αENaC immunolabel was readily detected in the CNT. However, αENaC label was not detected in any cells within the CCD of the collecting duct αENaC KO mice , which is consistent with the previous observation that αENaC is not expressed in the collecting duct of these mice [Because d in ICs , we explxloxcre)  , αENaC immunolabel was examined in 3 cell populations of the CCD. The first population did not express either the B1 subunit of the H+-ATPase or pendrin, which indicates they are principal cells. Many of these cells showed clear apical αENaC localization. The second cell population had distinct apical pendrin immunolabel and diffuse H+-ATPase label, consistent with either type B or non-A, non-B ICs, i.e. pendrin-positive ICs [+-ATPase, but negative for pendrin, which indicates type A ICs. In all intercalated cell populations, we observed diffuse, cytoplasmic αENaC immunolabel (inset). However, αENaC immunolabel was generally weak in the type A IC when compared with principal cells or type B ICs. Since type A IC immunostaining was often close to the limit of detection, we could not determine if αENaC is expressed at a significant level in any or in just a subset of these cells. Similar observations were made in kidney cryosections from wild type mice under basal conditions  and collecting duct specific αENaC null mice  that received aldosterone for 7 days (Treatment #2). As shown  and their floxed αENaC control littermates . As shown Fig. , in bothWhile serum electrolytes and arterial blood gases were similar in the NaCl-restricted Liddle’s and wild type mice Table , multipl− absorption in mouse CCD. To answer this question, we measured Cl− absorption (JCl) in CCDs from age- and sex-matched aldosterone-treated pendrin null and wild type pendrin mice (Treatment #2) that expressed either wild type ENaC  or that were homozygous for the Liddle’s mutation . Figure − absorption is greater in the Liddle’s than in wild type mice. However, pendrin gene ablation produced a similar change in Cl− absorption, JCl, in CCDs from both Liddle’s mice and in mice that express wild type ENaC. As shown  [The present and previous studies show that αENaC gene ablation reduces , while i#2 & #3) , 24, 27.− absorption in the mouse CCD by changing the benzamil-sensitive component of Cl− absorption, independently of pendrin. While pendrin markedly regulates ENaC abundance, subcellular distribution, and function either in NaCl-restricted or in aldosterone-treated mice, ENaC does not have a similar effect on pendrin under the same treatment conditions.We conclude that αENaC and pendrin co-localize to type B and non-A, non-B ICs in rat and mouse kidney, although the physiological role of αENaC within intercalated cells, remains to be determined. ENaC modulates Cl


Pancreatic cancer remains one of the deadliest cancer types. It is usually characterized by high resistance to chemotherapy. However, cancer-targeted drugs, such as sunitinib, recently have shown beneficial effects in pancreatic in vitro and in vivo models. Therefore, we chose to study a series of sunitinib derivatives developed by us, that were proven to be promising compounds for cancer treatment. The aim of our research was to evaluate the anticancer activity of sunitinib derivatives in human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 under normoxia and hypoxia. The effect on cell viability was determined by the MTT assay. The compound effect on cell colony formation and growth was established by clonogenic assay and the activity on cell migration was estimated using a ‘wound healing’ assay. Six out of 17 tested compounds at 1 µM after 72 h of incubation reduced cell viability by 90% and were more active than sunitinib. Compounds for more detailed experiments were chosen based on their activity and selectivity towards cancer cells compared to fibroblasts. The most promising compound EMAC4001 was 24 and 35 times more active than sunitinib against MIA PaCa-2 cells, and 36 to 47 times more active against the PANC-1 cell line in normoxia and hypoxia. It also inhibited MIA PaCa-2 and PANC-1 cell colony formation. Four tested compounds inhibited MIA PaCa-2 and PANC-1 cell migration under hypoxia, but none was more active than sunitinib. In conclusion, sunitinib derivatives possess anticancer activity in human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cell lines, and they are promising for further research. Cancer is one of the leading causes of death worldwide. According to the International Agency for Research on Cancer, in 2020 there were over 19 million new cases worldwide and almost 10 million deaths related to oncological diseases . Th. Th35]. 50) values were calculated for the most active and selective compounds EMAC4001, 4006, 4008, 40017 in normoxia and hypoxia  and 100 ± 13 and 80 ± 5 nM (in hypoxia). EMAC4008 showed statistically significant differences in its effects on MIA PaCa-2 and PANC-1 under normoxia and hypoxia conditions. Sunitinib possessed the lowest antiproliferative activity . All tested compounds inhibited cell viability statistically significantly compared to sunitinib C. The mo50 value was 9 μM), while the other pancreatic cancer cell line AsPC-1 cell line was the most resistant to imatinib  [No statistically significant difference was observed when comparing the effects of compounds between cell lines. Compound EMAC4006 and sunitinib inhibited more efficiently the cell viability of the MIA PaCa-2 cell line while compounds EMAC4001 and 4008 were more active in the PANC-1 cell line. PANC-1 cells are characterized by higher aggressiveness, therefore the greater inhibitory effect of the test compounds against this line is very important. Also, compound EMAC4008 was more active against both cell lines under hypoxia conditions. In contrast, sunitinib was more active under normoxia than hypoxia conditions. Another tyrosine kinase inhibitor imatinib showed the highest activity in the MIA PaCa-2 cell line (ECs 20 μM) . Gefitins 20 μM) . Meanwhi50 values, to avoid the possible misinterpretation of other effects in clonogenic and migration assays due to different possible effects on cell viability.For further research, we chose to use the compound concentrations equal to 10% and 90% from their calculated ECMIA PaCa-2 formed larger colonies compared to the PANC-1 cell line. Also, MIA PaCa-2 and PANC-1 cell colonies were smaller under hypoxia than under normoxia conditions A,B. 50 of EMAC4001 under normoxia and hypoxia conditions compared to sunitinib.In the clonogenic assay, the most active compound was EMAC4001. There were less of MIA PaCa-2 and PANC-1 cell colonies and they were smaller in the presence of 90% of EC50 reduced the number of PANC-1 cell colonies compared to the control under normoxia  (except for the effect of sunitinib at a lower concentration on the MIA PaCa-2 line under normoxia), statistically significantly inhibited cell migration of both lines under normoxia and hypoxia conditions. After 72 h, the ‘wound’ areas in MIA PaCa-2 and PANC-1 cell monolayers grown under hypoxia conditions were 58.90 ± 6.61 and 68.81 ± 5.24%, respectively  and published earlier . Sunitin2.The human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cell lines were obtained from the American Type Culture. Collection . MIA PaCa-2 and PANC-1 were cultured in Dulbecco’s Modified Eagle’s GlutaMAX medium . Medium was supplemented with 10% foetal bovine serum (Gibco) 1% antibiotic solution . Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO50 values that represent the concentration of a compound causing a 50% reduction of cancer cell metabolic activity have been calculated using the Hill equation. When determining the EC50 values of the compounds under hypoxic conditions, the growth medium was supplemented with 100 µM of CoCl2 solution .Cell viability was tested using MTT -2,5-diphenyltetrazolium bromide; Sigma-Aldrich Co.) assay, as described previously  Briefly,2 at 37 °C for 1 day. Then the monolayers were scratched with a sterile 100 μL pipette plastic tip in the centre of the well. The old cell medium is aspirated, the wells are rinsed with 400 μL phosphate-buffered saline . Then fresh medium containing compound concentrations representing 10% and 90% of the calculated EC50 value was added. Photos of ‘wounds’ were taken every 24 h for 3 days and the compound effect was evaluated by measuring the size of the “wound” area. Images were analysed by TScratch software .Cell migration was tested by the ‘wound’ healing assay, as described elsewhere . Briefly50 value. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C and grown for 8 days. After incubation, the cells were rinsed with PBS and fixed with 10% formaldehyde, methanol-free  solution for 20 min. Then the cells were rinsed with PBS two more times, and cell colonies were stained with 0.1% aqueous crystal violet solution (Sigma-Aldrich Co.) for 20 min, followed by rinses with sterile deionized water to remove the excess dye. Photos of colonies were taken using the G:BOX gel documentation system  and Genesys software . The number of colonies and their area were calculated using GeneTools colony and cell counting software  according to the manufacturer’s instructions. Cell colony area and number in control groups were normalized to 100%. The percentage of drug-treated colony area and number were compared to the control.Compound effect on cell colony formation and growth was established by clonogenic assay, as previously published . Brieflyp < 0.05.All experiments were repeated at least three times, calculating the mean and standard deviation. The data were processed using Microsoft Office Excel 2019 software . The level of statistical significance was set at In summary, it can be stated that the effects of the compounds in normoxia and hypoxia conditions in MIA PaCa-2 and PANC-1 cell lines are different. EMAC4001 possessed the strongest antiproliferative effect in tested pancreatic adenocarcinoma cell lines and was the most selective compound in MIA PaCa-2 and PANC-1 cell lines. All sunitinib derivatives at higher concentrations (except for compounds EMAC4008 and 4017 in the MIA PaCa-2 line), under normoxia conditions, showed an MIA PaCa-2 and PANC-1 cell colony formation inhibiting effect. EMAC4001 and sunitinib at a higher concentration significantly reduced the number and area of colonies in both cell lines under normoxia and hypoxia. Therefore, they could be suitable for the therapy of more aggressive, surviving cells and work in the presence of tissue hypoxia. Hypoxia is the main biological factor that causes tumour resistance to chemotherapy, therefore the stronger effect of compounds in hypoxia is very important. When comparing the effects of the compounds with sunitinib, none of the sunitinib derivatives inhibited cell migration more than the parent compound. Therefore, it could be hypothesized that sunitinib analogues would be more suitable for primary, non-metastatic pancreatic cancer treatment.


Rauwolfia tetraphylla is utilized by South Indian tribes to treat various medical ailments, although its cytotoxicity action has not been studied. As a result, the emphasis of the current investigation is on the anticancer activity ofRauvolfia tetraphylla extracts on MCF-7 breast cancer cell lines, as well as their effects on the levels of gene expression for BCl2 and TGF. The study found that the anticancer activity of R. tetraphylla extract demonstrated significant cytotoxic activityagainst MCF-7 breast cancer cell lines. Because of this, its anticancer effect may be caused by apoptosis, which is caused by DNA breaking and is helped by active phytochemicals like alkaloids, phenols, and flavonoids in the extracts. It also promotes apoptosisby altering Bcl-2 and TGF expression levels. The present study suggests using R. tetraphylla extract as an anticancer agent in traditional medicine.The medicinal herb  Rauwolfia tetraphylla L. (Family: Apocynaceae) is a tiny tree/shrub that grows to amaximum height of 6 feet. The tree has distinctive leaves that are generally medium to dark green in hue and whorled. Moreover, these trees are common in groups of four equal-sized plants per leaf node. These trees are also prevalent in India and are ofimportance owing to the distinctive indole alkaloids they contain [R. tetraphylla plant is commonly used by South-Indian tribes to treat many medical conditions. Many essential plant components, includingroots, stems, bark, and leaves, are employed to extract and isolate the chemical elements [R. tetraphylla has significant pharmacological action, making it useful in the treatment of a broad range of human healthissues via antimicrobial [Rauvolfia tetraphylla extracts on MCF-7 breast cancer cell lines and their impacts on BCl2 and TGF gene expression levels.Medicinal plants are vital to the worldwide survival of both human and animal populations. They are an abundant source of phytochemicals that may be utilized to treat various human illnesses ,2. In I contain . The R. elements . R. tetricrobial , anti-inicrobial , anti-caicrobial , and anticrobial activityicrobial . Availabicrobial , 15. Eveicrobial . These fWe used materials acquired from Gibco (Canada) for this experiment, including trypsin-EDTA, foetal bovine serum (FBS), antibiotics/antimycotics, DMEM, and PBS. Both the JC-1  andthe real-time PCR kit (MESA Green) were obtained from Invitrogen, USA. Pure, analytical-grade chemicals were employed at every step of the process.Rauvolfia tetraphylla were extracted with 70% ethanol using Soxhlet equipment. The extract was further filtered using Whatman No. 1 filter paper. The solvent was evaporated at low pressure using rotary evaporator equipment toget a viscous mass, which was then kept at 4°C until use.The bioactive components of The MCF-7 cell line was obtained from the National Center for Cell Science (NCCS) in Pune, India, and grown according to the instructions supplied. MCF-7 cells were cultured in MEM with 10% FBS at 37 degrees Celsius in a 5% CO2 incubator.R. tetraphyllaextracts and incubated for 24 hours at 37 °C in a 5% humidified CO2 incubator. Following that, 3--2,5-diphenyltetrazolium bromide (MTT) was added to each well, and the incubation was extended for 4 hours at 37°C. The cells wereresuspended in 200 µl dimethyl sulfoxide (DMSO) to dissolve the formazan produced by MTT. The optical density (OD) of the solution was measured using a spectrometer at 570 nm. The trials were carried out three times separately. Each replicate group's meanoptical density (OD) ±SD was computed.The human breast cancer cell line MCF-7 cell was seeded in 96-well plates at a density of 5x105 cells/well and allowed to adhere to the well overnight. After incubation, cultured cells were stimulated in triplicate with different doses of The inhibitory rate of cell growth was calculated using the equation:% Growth inhibition = (1 - OD extract treated)/OD negative control x 100.Total RNA was extracted from treated cell cultures using Trizol reagent (Sigma). Total RNA (2 µg) from each sample was reverse transcribed according to the manufacturer's procedure using a commercial Superscript III first-strand cDNA synthesis kit. For the BCL2 and TGF genes, real-time PCR was performed on an MX3000p PCR machine . MESA Green PCR Master Mix was used for the reaction  Eurogentec, Inc., USAMelting curve analysis was used to assess the specificity of the amplification product for each primer combination. The data was processed using the comparative CT approach, and the fold change was determined using the 2CT method given by Schmittgen andLivak (2008) with CFX Manager Version 2.1 .Data were presented as the means ±SD of three separate studies that were each carried out in triplicate. A one-way analysis of variance was used for the statistical analysis and a value of p<0.05 was used to indicate that the result was statisticallysignificant.R. tetraphylla, used in traditional medicine in different parts of South India to treat health problems, was looked at to find new drugs that might be better for treating cancer [R. tetraphylla were previously found to have no significant cytotoxicity against five human cancer cell lines, all with an IC50>40 µM [R. tetraphylla extract. The levels of expression of apoptosis-related genes, including Bcl-2 and TGF were analyzed,and it was shown that mRNA played a vital role in the process of apoptosis. The measurement of mRNA levels was carried out with the help of one step RT-PCR MESA green mix quantitative real time reverse transcription PCR. Compared with normal MCF-7 cell lines,the mRNA level of Bcl-2 in R. tetraphylla was shown to be considerably (p<0.01) up regulated. Compared with expression levels in normal MCF7 cell lines, the level of TGF produced by R. tetraphylla is considerably (p<0.001) lower lower.The mostne MCF-7 . The antne MCF-7 . There ane MCF-7 ,30. BaseR. tetraphylla extract demonstrated significant cytotoxic activity against MCF-7 breast cancer cell lines. As a result, the mechanism of its anti-cancer effect may be apoptosis produced by DNAbreakage, which is aided by active phytochemicals in the extracts, such as alkaloids, phenols, and flavonoids. It also promotes apoptosis by altering Bcl-2 and TGF expression levels. The present study warrants using R. tetraphylla extract as an anti-canceragent in traditional medicine. The present study concluded that the anti-cancer activity of


Radioresistance is a major cause of radiotherapy failure among patients with cervical cancer (CC), the fourth most common cause of cancer mortality in women worldwide. Traditional CC cell lines lose intra-tumoral heterogeneity, posing a challenge for radioresistance research. Meanwhile, conditional reprogramming (CR) maintains intra-tumoral heterogeneity and complexity, as well as the genomic and clinical characteristics of original cells and tissues. Three radioresistant and two radiosensitive primary CC cell lines were developed under CR conditions from patient specimens, and their characteristics were verified via immunofluorescence, growth kinetics, clone forming assay, xenografting, and immunohistochemistry. The CR cell lines had homogenous characteristics with original tumor tissues and maintained radiosensitivity in vitro and in vivo, while also maintaining intra-tumoral heterogeneity according to single-cell RNA sequencing analysis. Upon further investigation, 20.83% of cells in radioresistant CR cell lines aggregated in the G2/M cell cycle phase, which is sensitive to radiation, compared to 38.1% of cells in radiosensitive CR cell lines. This study established three radioresistant and two radiosensitive CC cell lines through CR, which will benefit further research investigating radiosensitivity in CC. Our present study may provide an ideal model for research on development of radioresistance and potential therapeutic targets in CC.The online version contains supplementary material available at 10.1007/s13577-023-00882-x. Cervical cancer (CC) is the fourth most common malignancy among women, accounting for approximately 7% of cancer cases , 2. AlthMost CC researches are based on traditional, established cell lines, such as SiHa, C33A, and ME180 etc. which are homologous in both morphology and phenotype and proliferate easily in vitro. However, loss of intra-tumoral heterogeneity is the main obstacle in developing drugs, including radiosensitizers, and research investigating radioresistance . A compaConditional reprogramming (CR) methods have been used to culture tumors from patients with CC with the support of feeder cells and conditional medium, enabling infinite proliferation in vitro . Whole-eThe development of high-throughput approaches, such as single-cell RNA sequencing (scRNA-seq), has enabled analysis of the molecular characteristics of individual cells in tumors. Nevertheless, the transcriptional spectrum of radioresistant and radiosensitive CC cells has not yet been investigated. The goal of current study was to establish radioresistant and radiosensitive CR cell lines from CC specimens and verified their characteristics in vitro and in vivo. For the first time, this study proclaims the molecular characteristics and biological changes of radioresistant and radiosensitive CC cells via scRNA-seq, providing a greater understanding of radiosensitivity that will benefit predictions of prognosis and radiotherapy response.Three radioresistant CC specimens were collected from local recurrent lesions of patients who received radiotherapy within 12 months and without re-irradiation or other treatments. Two radiosensitive CC specimens were collected from patients at diagnosis who achieved clinical remission after radiotherapy and remained disease-free survival for at least 12 months.2 incubator. All experiments were performed using mycoplasma-free cells. After reaching 80% confluence, the primary cells were passaged with 0.02% EDTA  and 0.05% trypsin/EDTA  consequently.CC tumor tissues were excised and cell suspensions were prepared following the protocol described by Liu et al. . BrieflyComplete F medium was prepared using DMEM , 10% fetal bovine serum , 1% 100 × Pen-Strep , 1% 100 × L-glutamine , 5 µg/mL insulin , 250 ng/mL amphotericin B , 10 µg/mL gentamicin , 0.1 nM cholera toxin , 0.125 ng/mL EGF , 25 ng/mL hydrocortisone  and 10 µM ROCK inhibitor Y-27632 .4 cells were seeded onto a 6-well plate in triplicate, and cell numbers were counted at 1, 3, 5, and 7 days. The growth rate and population doubling time were calculated from the growth curves.Cellular morphology was examined and photographed using a phase-contrast microscope . The population doubling times were calculated for cells in early passages. Briefly, 1 × 10Equal numbers of cells were plated in triplicate onto 6-well plates for 24 h, then received radiotherapy of 0, 2, 4, 6, 8 Gy. The plates were incubated at 37 °C for 2–3 weeks. Then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with 0.05% crystal violet. Colonies with > 50 cells were counted and photographed.Genomic DNA from primary cell lines and corresponding peripheral blood samples of patients was isolated using the Tissue DNA Kit . Matching comparison of DNA fingerprinting including a total of 21 STR loci  was conducted between peripheral blood samples and CR cell lines, as well as between the CR cell lines and data for traditional cell lines in the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Japanese Collection of Research Bioresources (JCRB) and RIKEN databases.6 cell suspensions which were prepared in 100 μL DMEM were conducted on left flank of the hindleg of anesthetized female mice. Injected mice were observed and the tumors were examined every three days. IHC straining was performed following the general procedure using the IHC kit .The animal research was performed under the NIH Guidelines for the Care and Use of Laboratory Animals and supervised by the Animal Care Committee of Tongji Medical College. In vivo primary cell lines were injected into 4–6 weeks old BALB/c nude mice . Briefly, subcutaneous injections of 5 × 104 cells were seeded on a chamber slide  in a 24-well plate for 24 h, followed by washing 3 times with PBS before being fixed with 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100 for 15 min, the cells were incubated with 5% bovine serum albumin (BSA) for 1 h at room temperature. Then cells were covered with primary antibodies against CK5 , Ki67 , p63 , and p16 , respectively, overnight at 4 °C. After 3 times washes, the secondary antibody  was added on the slides for 1 h. Nuclei were stained with DAPI . Chamber slides were analyzed and imaged by fluorescence microscope .A total of 5 × 10Raw disembarkation data quality statistics, cell ranger, cell refiltration, dimensionality reduction and clustering, differential genes and functional enrichment analysis, pseudo-time trajectory analysis, gene set variation analysis, CNV evaluation and transcriptional factor prediction were described in the Supplementary Methods .p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Results were represented as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Data shown were representatives of at least three independent experiments, which showed similar results.Statistical significance was determined by the Student’s t-test or two-way ANOVA using GraphPad Prism 7.0 software , and presented as *The primary radioresistant CR cell lines  4, UCR7, and UCR8) and radiosensitive CR cell lines  14, and UCS19) formed cancer nests when cultured with irradiated feeder cells . However, the STR loci in the CR cell lines differed from those of traditional cell lines reported in the ATCC, DSMZ, JCRB, and RIKEN databases.Next, xenografts of CR cell lines were established in mice and their tumorigenicity was verified. The xenografts kept the radiosensitivity of PDTs after irradiation Fig. a, b, exhTo investigate the crucial oncogenes or pathways associated with radiosensitivity in CC, CR cell lines UCR4 and UCS19 were selected to conduct scRNA-seq using 10 × Genomics Chromium platform. High-dimensional data were visualized into low dimensional space by t-distributed stochastic neighbor embedding (t-SNE). Cells with similar characteristics were classified into a cluster according to the algorithm of t-SNE, and the identified clusters were displayed. A mixture of human CR cell lines and murine feeder cells were captured Fig. a, c. A tIn general, cells in different cell cycle phases exhibit different radiosensitivity. Previous studies have shown that cells were most sensitive to irradiation during mitosis and in the G2 phase, less sensitive in the G1 phase and least sensitive in the latter S phase . The CR Next, the scRNA-seq results for UCR4 and UCS19 were combined to better understand the radiosensitivity of CC cells, yielding 13 cell clusters using the graph-based clustering method  were addressed in state 1, whereas the majority of radiosensitive cell clusters were placed in state 4. Branched expression analysis modeling (BEAM) indicated that cell cluster 8 was at one end of the pseudotemporal trajectory, while cell cluster 9 was located at the other end , these cell lines may serve as a promising platform for cancer research, including studies exploring cancer biology, high-throughput drug screening, personalized treatment, and biobanking repositories. Radioresistant cells are in G1 and S phases and increase the FoxO signaling and oxidative stress pathway to support the cells after irradiation. This study infers that USP7 can be the potential radiosensitization targets after careful scientific researches in the future.Supplementary file1 (PDF 1094 KB)Supplementary file3 (XLSX 32 KB)Below is the link to the electronic supplementary material.


JCI, Li, Wang, et al. report that T cell Stat3 deficiency can extricate GVL effects from GVHD through tissue-specific programmed death-ligand 1/programmed cell death protein 1–dependent (PD-L1/PD-1-dependent) bioenergetic alterations that blunt harmful T cell effects in GVHD target organs, while preserving their beneficial antitumor activity in lymphohematopoietic tissues.Allogeneic hematopoietic cell transplantation can cure patients with high-risk leukemia through graft-versus-leukemia (GVL) effects, the process by which malignant leukemic cells are cleared by donor-derived immune cells from the graft. The problem of harnessing GVL effects while controlling inflammation and host-organ damage linked with graft-versus-host disease (GVHD) has been the most formidable hurdle facing allogeneic hematopoietic cell transplantation. This powerful, curative-intent therapy remains among the most toxic treatments in the hematologist’s armamentarium due to the combined risks of GVHD-related morbidity, infections, and leukemia relapse. In this issue of the  In patients with blood cancers, the goal of allogeneic hematopoietic cell transplantation is to unleash curative graft-versus-leukemia (GVL) effects while minimizing morbidity from graft-versus-host disease (GVHD) . It has Stat3 inactivation in CD4+ T cells prevented chronic sclerodermatous GVHD in mice and limited donor T cell accumulation in vivo while expanding Tregs and decreasing numbers of inflammatory Th17 cells  to protect from acute GVHD  signaling. Stat3 inactivation in T cells enhanced PD-L1/PD-1–mediated inhibition of metabolic pathways in alloreactive T cells, thus interfering with metabolic reprogramming and leading to T cell dysfunction specifically in GVHD target organs  in a PD-1–dependent manner (Stat3-deficient alloreactive T cells were associated with decreased Myc expression and activity, decreased ATP production, and evidence of T cell dysfunction in GVHD target tissues (Stat3 deficiency intersects with PD-1 signaling or its downstream consequences in T cells.Li, Wang, et al. showed that GVHD prevention achieved by  tissues . Stat3 dt manner . The antt manner . Consist tissues . Yet it Stat3-deficient T cell recipients, while GVL was largely preserved. Indeed, genetic or pharmacologic inhibition of PD-L1/PD-1 signaling erased GVHD protection from Stat3 inactivation (Stat3-deficient T cell metabolism between GVHD target organs and lymphohematopoietic tissue. These observations fit with work from others identifying the importance of tissue-resident, locally maintained donor-derived T cells in GVHD (PD-1 blockade can accelerate GVHD in mice and humans , 10. Thetivation . PD-L1/P in GVHD .Stat3 deletion should also be considered as part of evolving work on metabolic control of GVHD. While others focused on glycolysis or other bioenergetic pathways in GVHD (Stat3 loss (Stat3-deficient T cells showed decreased glutathione/Myc pathway activity and ATP production in the liver, but not in the spleen. These findings should prompt caution in the interpretation of T cell metabolic findings when assessed only in lymphoid organs rather than also in target tissues.The tissue-specific bioenergetic consequences of  in GVHD , 15, Li,at3 loss . SpecifiStat3 deficiency, Li, Wang, et al. found no impact of Treg depletion on GVHD severity or survival in their model (Stat3 deficiency in a mouse model of chronic sclerodermatous GVHD, where anti-CD25 treatment resulted in delayed, organ-specific GVHD in recipients of Stat3-deficient T cells (Stat3 deficiency’s protection from GVHD based on destabilization of natural Tregs and reduced iTreg differentiation from naive CD4+ T cells (Despite prior findings suggesting a role for Tregs in GVHD protection upon T cell ir model . Prior wStat3 deficiency, an important future direction should include identifying key ligands and receptors upstream of STAT3 signaling in GVHD. In past work, these authors speculated that IL-6 and IL-21 could act upstream of STAT3 signaling in pathogenic CXCR5–PD-1hi helper T cells implicated in chronic GVHD pathogenesis (While the Li, Wang, et al. report explored metabolic consequences of ogenesis . It is uogenesis . Howeverogenesis . Thus, iGoing beyond genetics as a discovery approach, Li, Wang, et al. explored a pharmacological strategy of targeted STAT3 degradation with translational potential . They teStat3 deficiency protected mice from GVHD while maintaining potent GVL responses (Stat3 deficiency relied on PD-L1/PD-1 signaling. PD-L1 expression in GVHD target tissues conferred protection from GVHD that was not shared in lymphohematopoietic tissue, where donor T cells are likely to encounter hematological neoplasms. PD-1 signaling in Stat3-deficient T cells resulted in inhibition of the glutathione/Myc pathway, culminating in T cell dysfunction. The upstream signals and downstream pathways connecting STAT3 and PD-1 remain subject to further investigation. Furthermore, the role of Tregs appears unresolved given divergent data between Li, Wang, et al. and prior work about the effects of anti-CD25 treatment on GVHD after transplantation with Stat3-deficient T cells. Altogether, Li, Wang, et al. propose STAT3 degradation as an attractive strategy for controlling GVHD while maintaining potent GVL activity as well as an interesting concept in which tissue-specific T cell dysfunction explains the distinct effects of STAT3 loss on harmful and beneficial T cell responses (T cell esponses . The relesponses .


Snd1 from the mouse β cell lines, which revealed numerous differentially expressed genes linked to insulin secretion and cell proliferation, including limited expression of Glp1r. We observed Snd1 deficient β cell lines had reduced cell expansion rates, GLP1R protein levels, and limited cAMP accumulation under stimulatory conditions, and further show that acute ablation of Snd1 impaired insulin secretion in rodent and human β cell lines. Lastly, we discovered that PDX1:SND1 interactions were profoundly reduced in human β cells from donors with type 2 diabetes (T2D). These observations suggest the PDX1:SND1 complex formation is critical for controlling a subset of genes important for β cell function and is targeted in diabetes pathogenesis.The pancreatic β cell synthesizes, packages, and secretes insulin in response to glucose-stimulation to maintain blood glucose homeostasis. Under diabetic conditions, a subset of β cells fail and lose expression of key transcription factors (TFs) required for insulin secretion. Among these TFs is Pancreatic and duodenal homeobox 1 (PDX1), which recruits a unique subset of transcriptional coregulators to modulate its activity. Here we describe a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), which has been shown to facilitate protein-protein interactions and transcriptional control through diverse mechanisms in a variety of tissues. PDX1:SND1 interactions were confirmed in rodent β cell lines, mouse islets, and human islets. Utilizing CRISPR-Cas9 gene editing technology, we deleted   Forge physical connections between DNA-bound TFs and the broader transcriptional machinery via protein-protein interactions2, or 3. Covalently alter histone tails through processes such as acetylation, methylation, glycosylation, phosphorylation, ubiquitination, and sumoylation3. Each of these functions is executed in a gene-specific manner, entailing interactions with DNA-bound TFs that hinge on cellular cues for their recruitment and activity.Transcriptional coregulators constitute a pivotal component of gene regulation within eukaryotic systems. Their precise recruitment to DNA-bound transcription factors (TFs) elicits either positive or negative impacts on gene transcription. Varied classes of coregulators operate to: 1. Modify chromatin structure, thereby rendering it accessible or restrictive to other proteins through both enzymatic and non-enzymatic mechanisms4–6. For example, PDX1 is one of the diabetes-associated genes in which homozygous mutant humans exhibit pancreatic agenesis7, while heterozygotes develop early-onset diabetes consistent with both a developmental and functional role for PDX18–11. Moreover, conditional removal of Pdx1 from mature mouse β cells results in severe glucose dyshomeostasis that stems from rapid deterioration of gene expression programs essential for maintaining β cell function and identity4. Inactivation of islet-enriched TFs is associated with and Type 2 diabetes (T2D) pathogenesis. For example, gene expression and immunofluorescence analyses revealed that expression of PDX1, NKX6.1 and MAFA, a sensitive PDX1 gene target are reduced in islets from human donors with T1D and long standing T2D12. Given the influential role of Pdx1 in islet ontogeny and β cell function, as well as the control Pdx1 has on the transcriptional properties to maintain β cell identity, understanding ways in which Pdx1 transcriptional activity is modulated is paramount to define its role in diabetes pathogenesis.Pdx1 is one of the most influential TFs in the pancreas, as it plays a central role in pancreatogenesis, islet β cell development and function and maintenance of β cell identityin vitro revealed Pdx1 interacts with a diverse group of chromatin modifiers, including the Ruvbl1/2 DNA helicase, ATP-dependent SWI/SNF chromatin remodeling complex, the chromodomain helicase DNA-binding protein 4 (CHD4) subunit of the Nucleosome Remodeling and Deacetylase (NuRD) complex13, and the Staphylococcal Nuclease And Tudor Domain Containing 1 (SND1) coregulator ). The SWI/SNF chromatin remodeling complex was shown to control pancreatic progenitor cell proliferation and regulate insulin production by permitting PDX1 association with the Ins2 enhancer14. Furthermore, the CHD4 subunit of the NuRD complex was found to regulate glucose homeostasis in vivo and MafA expression by controlling MafA Region 3 enhancer accessibility15. Here our focus is on interrogating the role of the SND1 coregulator on controlling gene expression and β cell function in vitro.Previous work from our group and others has demonstrated the importance of PDX1-associated coregulators in modulation of its transcriptional activity in pancreas development and the mature β cell. An unbiased mass spectrometry screen 17. SND1 has been shown to modulate the transcriptional activity of the STAT5 and STAT6 TFs by controlling inflammation induced gene expression responses in B-lymphocytes19. In preadipocytes, PPARγ activity is regulated by Snd1 to promote adipogenesis20. In these settings, Snd1 acts as a coactivator where it bridges TFs with components of the RNA polymerase II machinery. SND1 has also been shown to govern chromatin accessibility in ovarian cancer cell lines through recruitment of histone acetyltransferases p300 and GCN521. Additionally, SND1 has garnered attention as a metastatic cancer biomarker given its positive correlation with cancer progression22. Herein, we characterize the role of Snd1 in controlling β cell function. We demonstrate that PDX1 and SND1 interact in rodent and human β cells, where reduced levels of Snd1 negatively influence insulin secretion. β cell expansion and gene targets critical for β cell function are markedly affected in cells deficient for SND1. Lastly, we reveal interactions between PDX1 and SND1 are reduced in T2D donor β cells and discuss the implications of our findings in relation to T2D pathogenesis.SND1 contains four repeated staphylococcal nuclease domains and a single Tudor domain, which comprises its endonuclease activity and ability to interact with individual proteins, coregulatory complexes and nucleic acids23.Rodent and human β cell lines: mouse βTC3 cells, mouse insulinoma 6 (MIN6) cells, rat INS-1 832/13 and human EndoC-βH1 cells (passage number 65–70 used) were cultured using protocols as previously described23. To generate lentivirus expressing Snd1 specific gRNAs, we transfected HEK2 93T cells with packaging plasmids pVSV-G, psPAX2 and puromycin-resistant single guide gRNA transfer vectors ) )  in Opti-Snd1 was performed as previously described23 using the following small interfering (si)RNA for rodent (INS-1 832/13) and human (EndoC-βH1): ON-TARGETplus mouse Snd1 , human SND1 , nontargeting control . After transfection, RNA and nuclear extract were collected at 48 hours for INS-1 832/13 cells and at 72 hours for EndoC-βH1 cells. The nuclear extracts were fractionated following the procedure previously described23. PVDF membranes were then probed with rabbit α-SND1 antibody  and mouse α-β-actin antibody . The LI-COR system was utilized in conjunction with secondary antibodies to capture images of the blots. The experiments were repeated at least three times, and ImageJ software was employed to quantitate the results.Knockdown of All animal studies were reviewed and approved by the Indiana University Institutional Animal Care and Use Committee. Mice were housed and cared for according to the Indiana University Laboratory Animal Resource Center and the Institutional Animal Care and Use Committee/Office of Animal Welfare Assurance standards and guidelines. Twelve-week-old male C57BL/6J mice (Jackson Laboratories) were used to isolate whole mouse pancreata. The pancreata were fixed in 4% (v/v) paraformaldehyde/PBS for 4 hours, washed, and then embedded in O.C.T. embedding medium. Cryosections of 6 μm thickness were obtained using a Leica Cryostat. Human pancreatic tissue sections were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD) .Table 2.To conduct co-immunoprecipitation experiments, 100 μg of either βTC3 or INS-1 832/13 nuclear extracts was combined with goat α-PDX1 antibody , α-SND1 antibody (PA5–40124), and either goat or rabbit IgG. The mixture was then incubated overnight at 4°C. The following day, Protein A/G PLUS-agarose was used to precipitate the antibody complexes, which were subsequently washed with PBS and boiled with SDS loading dye. The immunoprecipitants were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with Blocking Buffer . Membranes were probed with primary antibodies: goat α-Pdx1  and rabbit α-SND1 . For detection, LI-COR IRDye secondary antibodies were employed, and fluorometric scanning was carried out using an Odyssey CLx Imager. Finally, ImageJ software was utilized to quantify the band intensity.EndoC-βH1 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 24 hours after seeding, before blocking and adding primary antibodies. For mouse cryosections, tissue sections were washed and subjected to 1% SDS-mediated permeabilization prior to blocking and adding primary antibodies. For paraffin-embedded human pancreatic sections from nPOD donors, heat-activated antigen retrieval  was performed before blocking and adding primary antibodies.The proximity ligation assays (PLAs) were conducted following the guidelines provided by the manufacturer . The primary antibodies used for proximity ligation were goat α-Pdx1  and rabbit α-SND1 . Guinea pig α-insulin  and mouse α-proinsulin targeting human proinsulin  were utilized for tissue sections. The detection of insulin and human proinsulin was performed using Cy2 secondary fluorophores. Immunofluorescence Z-Stack images were acquired through the depth of the tissues/cells using a Zeiss LSM 800 confocal laser scanning scope and processed using ImageJ software. To quantify PLA signals in human tissue sections, we manually enumerated the signals from individual Z-stack images, then plotted the signal counts in relation to the total number of β cell nuclei quantitated.Snd1 KO cell lines with RNA integrity numbers ≥ 7.0 was used for library following cDNA preparation (Takara) with the Nextera XT DNA Library Prep Kit . Each library was quantified, and quality accessed by Qubit and Agilent Bioanalyzer with average size of the library insert ~ 300–400 basepairs. Libraries were denatured and neutralized before loading to the NovaSeq 6000 sequencer for 100b paired-end sequencing . Approximately 12–18 × 106 reads per library were generated. A Phred quality score (Q score) was used to measure the quality of sequencing with more than 95% of the sequencing reads reached Q30 . The generated FASTQ files were processed using the Genialis visual informatics platform . Raw and analyzed mRNA-Sequencing data sets have been deposited in GEO (accession number: GSE240993).One hundred nanograms of RNA from Control and Snd1 knock out MIN6 cells was carried out following the manufacturer’s instructions . Subsequently, cDNA synthesis was performed according to the manufacturer’s guidelines . Quantitative (q)PCR reactions were conducted using the gene primers listed in −ΔΔCT method was employed with 18s used as the normalization reference.RNA extraction from Control and 6 cells per well in a 6-well plate. After either 48 (INS-1 832/13) or 72 (EndoC-βH1) hours, cells were exposed to a baseline glucose solution containing 1 mM glucose for 1 hour. The solution was then removed, and the cells were treated with either 2.8 mM glucose (low) or 16.7 mM glucose (high) for 1 hour. Secretion media was collected, and acid/ethanol solution was added to the cells to collect content samples. Measurement of INS-1 832/13 and EndoC-βH1 cell secretion media were performed using human insulin ELISA since INS-1 832/13 cells contain a human insulin expression cassette24. The ELISA was conducted by the Translation Core at Indiana University School of Medicine. To ensure consistency, all secretion samples were normalized to insulin content and fold change calculated by dividing the percentage of secreted insulin at high glucose to the percentage of secreted insulin at low glucose for each experimental replicate.INS-1 832/13 and EndoC-βH1 cells treated with siControl and siSnd1/siSND1 were seeded at a density of 2 × 10Snd1 knockout cells for 60 minutes before fixation in 4% paraformaldehyde. Mouse monoclonal α-E-cadherin 1:500  antibodies were applied overnight at 4°C in PBS + 0.1% Triton + 1% BSA. Secondary Cy5 donkey α-mouse  antibodies were applied in the same buffer for 2 hours at room temperature. Images were acquired on a Zeiss LSM 800 confocal laser scanning scope and processed using ImageJ software. GLP1R surface expression was quantified vs. total E-cadherin expression and normalized against control cell lines.LUXendin551  was applied to MIN6 Snd1 knockout and control MIN6 cells. Briefly, 7500 cells/well were seeded into a 384-well plate and cells were incubated with 50 nM Exendin-4 in the presence of 16.7 mM glucose at 37°C in a humidified incubator with 5% CO2 for 30 minutes. Cell lysis and measurement of cAMP were carried out with a HTRF (Cisbio) assay according to the manufacturer’s instructions using a Molecular Devices SpectraMax iD5 plate reader. All assays were performed in the presence of 500 μM IBMX to inhibit phosphodiesterase activity.cAMP assays were performed in Snd1 knockout cell lines were used to prepare the cell suspension at a concentration of 5 × 104 cells/well in 100 µl culture medium. The suspensions were then seeded in 96-well plates (100 µl/well) and incubated at 37°C in a humidified incubator with 5% CO2 for required exposure time. Each day following, WST-1 reagent solution was added to each well and the plate is incubated at 37°C for 2 hours. After incubation, the absorbance is measured at 450 nm with a multiplate reader.MIN6 control and p < 0.05 was used to identify significant differences between groups. Each experiment was repeated a minimum of three times.To determine statistical significance, the two-tailed Student’s t-test and/or One-way ANOVA were employed. The data are presented as the mean ± SEM. A significance threshold of To validate the novel PDX1:SND1 interactions discovered through unbiased IP-mass spectrometry analysis using nuclear enriched extract from rodent pancreatic β cell lines ), we conducted co-immunoprecipitation (co-IP) experiments on non-crosslinked nuclear enriched extracts from two different rodent lines: βTC3 and INS-1 832/13 cells. For the co-IP, we employed PDX1 and SND1 antibodies to capture the complexes associated with the proteins and then immunoblotted for SND1 and PDX1. The results revealed that SND1 and PDX1 proteins are pulled-down in the reciprocal co-IP labels in comparison to the IgG controls, thereby confirming the interaction between these proteins .Figure To further validate the presence of PDX1:SND1 complex formation in β cell nuclei, we conducted proximity ligation assays (PLA) using α-SND1 and α-PDX1-specific antibodies in 12-week-old C57BL/6J mouse pancreatic tissue , human ESnd1 via siRNA in the rat INS-1 832/13 and human EndoC-βH1 β cell lines, which achieved 51 ± 31% (INS-1 832/13) or 50 ± 8.2% (EndoC-βH1) knockdown  in β cells, we knocked down Snd1, we utilized CRISPR/Cas9 gene-editing technology to delete Snd1 from MIN6 β cell lines. Previously generated Cas9 expressing MIN6 β cell lines23 were infected with lentivirus encoding gRNAs targeting exon 2 coding region or a non-coding intron region (used as control) of Snd1  .To generate tools to investigate the defect in GSIS observed in rodent and human β cell lines following reduction of  of Snd1 . FollowiSnd1 KO) . SequencSnd1 KO β cell lines, we performed unbiased mRNA sequencing and based differential gene expression on a two-fold cutoff and false discovery rate of < 0.05. With these parameters, we discovered 80 downregulated- and 79 upregulated-genes Glp1r, Crp, Rgs8, Rgs16, Sct, and Sp8) and upregulated genes   . Gene On2, Six2) , the latll lines . Interes and INS . We nextSND1 has been linked to cell proliferation in the context of human cancer progression and metastatic spread26. We followed up on our GO pathway analyses findings by performing a WST-1 assay for cell expansion. Control and Snd1 KO cells were seeded at the same density and the WST-1 reagent was added on sequential days to measure expansion. The cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases is measured as a proxy for the number of viable cells in the dish. Interestingly, we revealed that Snd1 KO lines have significantly blunted cell expansion capacity in comparison to control lines .Interestingly, 27. GLP-1, an incretin that targets GLP1R, has been shown to have several effects, including potentiating insulin secretion, inhibiting glucagon secretion, slowing gastric emptying, and promoting satiety28. Reagents for the detection of GLP1R protein in tissues are limited. Recently, GLP1R fluorescent antagonists (termed LUXendins) have been generated to efficiently label GLP1R on the surface of tissues in which it is expressed30. To evaluate changes in GLP1R protein levels, Snd1 KO and control β cell lines were treated with the fluorescent LUXendin 551 probe and counterstained with E-cadherin to mark the surface of the β cells. Quantitative analyses revealed that GLP1R levels are trending down in Snd1 KO cell lines .GLP1R is a class B G-protein-coupled receptor involved in glucose homeostasis and serves as a recent modality for treating T2D and obesity32. Increased cAMP levels play a crucial role in the potentiation of GSIS by enhancing insulin granule exocytosis and gene expression of insulin and other factors involved in insulin secretion33. Given the trending reduction of GLP1R protein in Snd1 KO cells  and the link of other genes differentially expressed to cAMP biosynthesis (Snd1 KO β cell lines upon high glucose (16.7 mM) in presence of GLP1R-agonist, Exendin-4. We observed a trending reduction in cAMP accumulation in the Snd1 KO cell lines as compared to control cell lines .Upon agonist binding to the GLP1R on the surface of pancreatic β cells, downstream signaling leads to activation of the enzyme adenylate cyclase, which converts ATP into cyclic adenosine monophosphate (cAMP)ynthesis , we meas23, indicating an important role for the PDX1:BRG1 and PDX1:CHD4 complex formation to maintain β cell function. To assess whether PDX1:SND1 demonstrate similar perturbed interactions, we utilized PLA on closely age-, BMI- and sex-matched non-diabetic and T2D human donor tissue sections procured from nPOD (Donor information in n = 4). We observed that a greater number of β cell nuclei were devoid of PLA signals and fewer β cell nuclei contained 1 and 2 PLA signals in T2D donor tissue compared to non-diabetic donor tissue Previously, our group and others have revealed that interactions between PDX1 and the coregulators BRG1 and CHD4 are reduced in human T2D donor β cellsin vivo14. We have also shown the importance of PDX1:CHD4 interactions in maintaining pancreatic β cell function in both in vitro23 and in vivo15 studies. Here we have defined an important role for SND1, a newly defined PDX1-interacting partner, in controlling a subset of genes critical for β cell function.Gene regulation by PDX1 (and other TFs) is principally coordinated through protein-protein interactions between DNA-bound TFs and recruited coregulators, which positively or negatively impact transcription. There is a glaring need to understand how PDX1 transcriptional activity is controlled by coregulators under physiological conditions in the β cell, as this will lead to a deeper understanding of its failure in T2D. In this regard, PDX1 was shown previously, and recently by our group, to interact with a diverse set of transcriptional coregulators in the β cell, several of which have been shown to modulate its activity. For example, the ATP-dependent SWI/SNF chromatin remodeling complex positively contributes to PDX1 by regulating gene expression of a subset of PDX1 target genes critical for β cell function and identity 34, scaffolding for protein-protein interactions35, nuclease functions, miRNA processing36, and transcription regulation37. Accordingly, with its diverse functional roles, SND1 protein is detected in multiple intracellular locations, including the endoplasmic reticulum38, stress granules39, lipid droplets40, and nuclei41. We have also observed this using the tools at our disposal, with SND1 being present in nuclear enriched extracts of β cell lines . We predict these defects are likely due to proliferation delays, since should SND1 be involved in restraining apoptosis in the β cell, Snd1 KO cell lines would have never thrived during expansion of initial single cell seeding to generate the lines. Our current study is limited to utilizing in vitro cell line model systems where Snd1 is transiently reduced using siRNAs or knocked out by CRISPR technology. Future efforts on the role of SND1 in β cell expansion will be addressed using in vivo model systems.Given the role of SND1 in pivotal biological processes like regulation of gene expression, cell cycle and overexpression in cancer setting feration . MoreoveSnd1 knockdown cell lines to secrete insulin under high glucose stimulatory conditions. We speculate this is likely a result of glucose processing and/or insulin exocytosis defects, as we did not observe changes in INSULIN processing enzymes in RNASeq datasets and INSULIN content is unchanged , further linking the gene expression changes following Snd1 deficiency to those functional aspects of β cell biology. The potential role of Snd1 in this pathway is especially relevant as exendin-4 has been utilized to improve glycemic outcomes in individuals with T2D. Interestingly, we observed some gene expression differences between Snd1 KO cell lines and siSND1 EndoC-βH1 cells. For example, we observed trending reductions in INS and MAFA genes in siSND1 EndoC-βH1 cells; however, these transcripts were unchanged in Snd1 KO cells and INSULIN content was unaltered in both cell lines  during T2D pathogenesis, prior to significant reductions in PDX1 protein levels, would be of significant value to understanding the mechanisms of β cell failure in T2D.T2D is a complex, polygenic disease with a heterogenous pathophysiology, associated with dysfunctional β cells β cells . As within vitro and in vivo and these interactions are negatively impacted in pathophysiological settings of T2D. These findings are the first to demonstrate that SND1 plays an essential role in maintaining β cell function and provide evidence that loss of PDX1:SND1 complex formation is a significant feature of diabetes pathophysiology. Future studies will be focused on dissecting the molecular mechanisms by which SND1 is able to regulate insulin secretion and proliferation in β cells using in vivo model systems.Overall, our results demonstrate a critical role of SND1 in controlling insulin secretion and modulating a subset of genes important for β cell function. We have discovered that PDX1 interacts with SND1 in the nuclei of pancreatic β cells both Click here for additional data file.


MS) state and p53 genotype. It shows that cell-cycle checkpoint, metabolism of proteins and RNA, signal transduction, and WNT signaling processes are significantly more active in MSI-High p53-WT cell lines. Conversely, MSI-High cell lines with a mutant (Mut) p53 gene showed hyperactivation of cell signaling, DNA repair, and immune-system processes. Several kinases were linked to these phenotypes, from which RIOK1 was selected for additional exploration. We also included the KRAS genotype in our analysis. Our results showed that RIOK1’s inhibition in CRC MSI-High cell lines was dependent on both the p53 and KRAS genotypes. Explicitly, Nintedanib showed relatively low cytotoxicity in MSI-High with both mutant p53 and KRAS (HCT-15) but no inhibition in p53 and KRAS WT (SW48) MSI-High cells. This trend was flipped in CRC MSI-High bearing opposite p53-KRAS genotypes , where observed cytotoxicity was more extensive compared to the p53-KRAS WT-WT or Mut-Mut cells, with HCT 116 (KRAS-Mut and p53-WT) being the most sensitive to RIOK1 inhibition. These results highlight the potential of our in silico computational approach to identify novel kinases in CRC sub-MSI-High populations as well as the importance of clinical genomics in determining drug potency.The vulnerabilities of cancer cells constitute a promising strategy for drug therapeutics. This paper integrates proteomics, bioinformatics, and cell genotype together with in vitro cell proliferation assays to identify key biological processes and potential novel kinases that could account, at least in part, for the clinical differences observed in colorectal cancer (CRC) patients. This study started by focusing on CRC cell lines stratified by their microsatellite ( Colorectal cancer (CRC) is the third-most-common cancer in Western countries and the second leading cause of cancer-associated death globally . CRC shoMicrosatellites (MS) are short DNA tandem repeats (1–6 nucleotides) that represent approximately 3% of the human genome. MS generation is believed to occur by DNA slippage during DNA replication and repair, resulting in repeating nucleotides being skipped or inserted. Microsatellite instability (MSI) occurs when mismatch repair (MMR) enzymes are inactive and tumors containing at least two of the five known MSI markers are referred to as MSI-High cell lines . In CRC,KRAS-mutated CRCs had a lower frequency in MSI-High  compared with KRAS-WT, though KRAS mutation was associated with higher tumor-cell turnover [Genomic-wise, CRC cell lines possess a high frequency of TP53 (60%) mutations. Dysregulation of the p53 tumor suppressor gene is one of the most frequent factors controlling the aggressive and metastatic features of CRC. Half of all colorectal cancers containing p53 mutations, appear to be more chemo-resistant and have a poorer prognosis than those with wildtype (WT) p53 ,11,12. Iturnover .A study of Bosnian colorectal cancer patients identified a potential link between alterations in certain tumor-suppressor genes and the MSI phenotype . SpecifiThis work integrates cell genotype, proteomics, bioinformatics, and in vitro cell proliferation studies to identify key biological processes and kinases that could account for the clinical differences observed between CRC patients. In silico analysis was done on 34 CRC cell lines stratified by their MS status (MSI and MSS) and p53 (wildtype and mutant) genotype with a focus on differences in pathways activation levels and kinase abundance. We hypothesized that targeting highly abundant kinases in MSI-High cell lines, which have specific vulnerabilities due to inactive MMR genes, might disrupt the delicate balance between cell proliferation and cell death, inducing apoptosis. The rationale for this approach is three-fold. First, some mutant p53 proteins lose their tumor-suppressor activity and gain oncogenic functions, providing advantages to survival . Second,To address this need, we investigated whether a novel SL partner could be identified in CRC cells. There are known differences in the activity level of signaling proteins in MSI-High p53-WT and MSI-High p53-Mut cell lines, and gene expression is altered in CRC cells with MSI compared to non-MSI . In our We tested CRC MSI-High (both p53/KRAS and WT/Mut) cell lines using Nintedanib, a known RIOK1 inhibitor, to validate this hypothesis. As a control, 5FU was used as it is a first-line treatment for colorectal cancer ,26. Our KRAS mutations confer the constitutive activation of this oncogene, stimulating cell proliferation, inducing autophagy, suppressing apoptosis, altering cell metabolism, changing cell motility and invasion, and modulating the tumor microenvironment [Mantovani et al. have suggested that mutant p53 proteins can favor cancer-cell survival and tumor progression by acting as homeostatic factors that sense and protect cancer cells from transformation-related stress stimuli, including DNA lesions, oxidative and proteotoxic stress, metabolic imbalance, interaction with the tumor microenvironment, and the immune system . Similarironment . These mironment . Our iniCRC cell lines can be divided based on MS status and p53 genotype: Colorectal tumors with MSI have distinctive features, including a tendency to arise in the proximal colon, lymphocytic infiltrate, and a poorly differentiated, mucinous, or signet ring appearance. While they have a slightly better prognosis with immune therapy than MSS colorectal tumors, they do not show improved survival in CRC stages II and III compared to MSI-High patients treated with chemotherapy . Based oUnique highly activated pathways exist in MSI-High p53-Mut cell lines: The pathways that are most active in MSI-High cell lines were then investigated using the ATLANTiC portal . Pathwayp < 0.01 or p < 0.05) levels of activation between the MSI-High p53-WT and MSI-High p53-Mut using a chi-squared analysis   is consiRecent publications have demonstrated an SL role for RIOK1 in methylthioadenosine phosphorylase (MTAP)-depleted cells and its overexpression in colon cancer cells was shown to promote cell proliferation in vitro in human CRC ,48. HoweKnown Kinase inhibitor drugs and their downstream pathways in p53-Mut MSI-High cell lines: Using data published by Frenjo, we generated a Venn diagram  that depThose nine drugs belong to six pathways ), further supporting our hypothesis that some tumor cells are reliant on RIOK1 expression (https://www.genome.jp/pathway/map05210 (accessed on 2 September 2021)) was analyzed to visualize how inhibition of RIOK1 might trigger novel pathways in MSI-High cell lines ) [Identification of MSI-High and cell genotype in CRC cell lines: MSI-High cell lines were identified using data from (1) the ExPasy database (r 2021)) ,23; (2) r 2021))  and knowr 2021)) . All datPathway analysis: Pathways that were shown to be highly activated with relative activity > 0.85 were extracted from the ATLANTiC data set . A cutof2 while SW48 cells were cultured in a humidified atmosphere without CO2.MSI-High Cell lines and culture conditions: SW48, LS411N, HCT 116, and HCT-15 cell lines were purchased from the American Type Culture Collection . SW48 cells were cultured in Leibovitz L-15 Medium, LS411N, and HCT-15 cells were cultured in RPMI-1640 Medium and HCT 116 cells were cultured in McCoy’s 5A Medium (ATCC). All media containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin  at 37 °C. LS411N, HCT 116, and HCT-15 cells were cultured in a humidified atmosphere containing 5% CON = 3, n = 6) and 0.1% Triton X was used as a positive control for cytotoxicity. Following 48 h or 72 h of incubation at 37 °C (48 h for SW48 and HCT-15 and 72 h for LS411N and HCT 116), WST-8 reagent was added to each well and the plate was placed in a 37 °C incubator for an additional 4 h. Optical density was measured at 450 nm on a BioTek microplate reader .Cell Viability by WST-8 (Cell Counting Kit-8) Assay: The cytotoxicity was measured using a cell viability assay  following the manufacturer’s instructions. Briefly, 100 µL cells per well were seeded into 96-well plates  and incubated for 24 h. Cells were treated with 5FU (150 µM) or Nintedanib  ; 5FU was stored as 50 mg/mL stock in dimethyl sulfoxide  at −20 °C. Nintedanib was obtained from Angene International Limited  and stored as 25 mg/mL stock in DMSO at −20 °C.Statistical analysis: Statistical analysis was performed using an unpaired t-test using GraphPad Prism version 9.1.2 for Windows, . Significance calculations of the biological processes between MSI-High p53 WT vs. Mut were calculated using a chi-square test .CRC is the third-most-common cancer in the world, arising from the colorectal epithelium as a result of the accumulation of genetic alterations. Two major mechanisms of genomic instability have been identified in sporadic CRC progression. The first, known as chromosomal instability (CIN), results from a series of genetic changes that involve the activation of oncogenes such as KRAS and inactivation of tumor suppressor genes such as p53, DCC/Smad4, and APC . The secKRAS-mutated CRCs had a lower frequency in MSI-High CRC  mutations and to a lesser extent KRAS mutations (49%). Seeking to provide the largest benefit to the CRC MSI-High population, we wanted to identify SL partner(s) that serve as an Achilles heel specifically for the p53 cancer cell line. Originally, we did not include the KRAS genotype in our hypothesis because RC . In addRC  p53 ,11. MoreThe rationale for clustering CRC cell lines based on their MSI-High phenotype is that these cell lines are known to exhibit clinical, pathological, and molecular characteristics which distinguish them from MSS cancer cell lines and p53 is a key regulator of cellular processes. Our analysis demonstrated that cell signaling, DNA repair, and immune system-related processes are significantly more abundant in MSI-High p53-Mut cell lines while cell-cycle checkpoint, metabolism of protein, metabolism of RNA, signal transduction, and WNT signaling were more pronounced in MSI-High p53-WT cell lines . Pathway analysis demonstrated that four specific kinases were upregulated in these pathways: TBK1, CAMK1, CHUK, and RIOK1 . SeveralTreating MSI-High CRC cell lines bearing p53 mutations with Nintedanib (a known RIOK1 inhibitor) was expected to show differences in efficacy. This expectation was derived from the significant difference in treatment response and patient outcome based on their p53 genotype . This obCRC MSI-High cell lines bearing opposite genotypes of p53 and KRAS, where one gene is WT and the other mutant  showed the highest sensitivity and the most significant reduction in cell proliferation . Contradicting what we found, recent results demonstrated that RIOK1 shRNA treated the LoVo and HCT 116 cell lines (both MSI-High p53-WT and KRAS-Mut (G13D) showed no difference in cell apoptosis between untreated and RIOK1 shRNA-treated cell lines . Of noteLarge data sets of human genomic, transcriptional, proteomic, and epigenetic data are readily accessible from tumor cells, which allows increased analysis of cell-cycle pathways heterogeneity. Understanding these heterogeneities makes the development of personalized treatments targeting specific proteins possible. As a result, in 2020, the FDA approved Keytruda (pembrolizumab) for intravenous injection for the first-line treatment of patients with unresectable or MSI-High or mismatch repair deficient colorectal cancer. This marks the first immunotherapy approved for this patient population as a first-line treatment and which is administered to patients without also giving chemotherapy. This trend opens new opportunities for MSI-High patients regardless of the precise molecular mechanisms underlying the effect of RIOK1 inhibition. Our data support RIOK1 as a potential target for MSI-High subpopulation (with either p53 or KRAS Mut) tumor therapy that currently has no effective treatment . RIO kinOur results demonstrate that systems approaches can uncover nonobvious, mechanistic bases for clinical observations that otherwise defy expert-level explanation. These findings validate the methods we have been using to identify new targets/kinases for anticancer therapies . RIOK1 r


Recently, dogs have been increasingly spaying at a young age to prevent mammary tumors. These CMTs require extensive local excision and exhibit a high probability of metastasis to the regional lymph nodes and lungs during malignancy. However, the molecular and biological mechanisms underlying CMT development have not been fully elucidated, and research in this area is limited. Therefore, in this study, we established new CMT cell lines by isolating cells from tumor tissues and investigated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), a target for human breast cancer. PIK3CA mutations were observed at a similar loci as in the human PIK3CA gene in half of all canine samples. Furthermore, we investigated whether alpelisib, a PIK3CA inhibitor approved by the U.S. Food and Drug Administration for human breast cancer treatment, along with fulvestrant, is effective for CMT treatment. Alpelisib exerted stronger anticancer effects on cell lines with PIK3CA mutations than on the wild-type cell lines. In conclusion, we established new CMT cell lines with PIK3CA mutations and confirmed the efficacy of alpelisib for CMT treatment  Despite the increasing interest in CMTs, CMT-derived cell lines have only recently been developed for laboratory research (Canine mammary tumors (CMTs) are commonly observed in female dogs, and approximately 50% of all cases are malignant . Mastectresearch –7, and aresearch –11. Baseresearch –5, no caresearch , 13. PI3research , 15. PI3research . Phospharesearch . In partresearch , 20. Notresearch , 22. Thein vitro anticancer effects of alpelisib on these cell lines.In addition to the importance of PIK3CA mutations in human breast cancer, various studies have shed light on their correlation with canine tumors –26. Tae-The normal canine Madin-Darby canine kidney (MDCK) cell line was purchased from the Korean Cell Line Bank . Dulbecco’s Modified Eagle’s Medium/nutrient mixture Ham’s F-12 (DMEM/F-12) was purchased from Corning . Fetal bovine serum (FBS) was purchased from Gibco . Trypsin-ethylenediaminetetraacetic acid (0.25%), phosphate-buffered saline (PBS), and penicillin/streptomycin were purchased from Welgene . Cell viability assay kit and Mycoplasma polymerase chain reaction (PCR) detection kit were purchased from Dogen  and Cellsafe , respectively. Alpelisib was purchased from Selleckchem .n = 5) and Haedeun Animal Medical Center (n = 7) after obtaining informed consent. After surgical removal, some tumor samples were fixed in formalin and embedded in paraffin (FFPE) for hematoxylin and eosin (H&E) staining. Veterinary pathologists conducted microscopic examinations to determine the tumor grade and evaluate cytological malignancy of the tumor cells. Other tumor sections were temporarily maintained in cold DMEM/F-12 for cell isolation.A CMT cell line was established using a female canine clinically diagnosed with CMT. All surgically removed mammary tumor tissue samples were collected at the VIP Animal Medical Center  were generated via enzymatic dissociation. Tissue samples were washed with PBS, fragmented into small pieces, and dissociated via resuspension in DMEM/F-12 containing collagenase  for 4 h at 37°C in a humidified atmosphere containing 5% CO6 cells were harvested and lysed using a lysis buffer containing RNase A. After incubation at 37°C for 30 min, protein precipitation buffer was added to the cell lysate, followed by brief centrifugation. The supernatant was transferred to a new tube and an equal volume of isopropanol (2-propanol) was added. After centrifugation, the pellet was washed with 70% ethanol and air-dried for 15 min. DNA rehydration buffer was added, and the extracted genomic DNA samples were submitted to Macrogen Inc.  for Sanger sequencing. To examine the c.3140A > G (H1047R) mutation and c.1637A > C (Q546P) mutation, the following sequencing primer sets, originated from a previous publication , following the manufacturer’s instructions. Briefly, approximately 1 × 10lication , were us4 cells per well. After 24 h, each well was trypsinized, and the cells were microscopically counted for 6 days. Growth curves were plotted using GraphPad Prism , and the doubling time (DT) was calculated using an online tool.To assess their growth characteristics, cells were seeded in a 6-well plate at a density of 5 × 105 cells/mL. After overnight incubation at 37°C, the medium was replaced with serum-free DMEM/F-12, and cells were treated with alpelisib, which was dissolved in 1% final concentration of dimethyl sulfoxide (DMSO) at various concentrations . Cells were then incubated at 37°C for 24 h, 48 h, and 72 h. At the end of each treatment period, the medium was aspirated, and the EZ-cytox reagent (WST) was added to each well. After incubation at 37°C for 1 h, cell viability was measured at 450 nm using the LUMIstar Omega microplate reader . Half-maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism .To evaluate the cytotoxic effect of alpelisib on CMT cell lines, the cells were seeded in a 96-well plate at a density of 1 × 105 cells/mL. After overnight incubation at 37°C, the medium was replaced with serum-free DMEM/F-12, and cells were treated with alpelisib, which was dissolved in 0.1% final concentration of DMSO at various concentrations . Then, the cells were incubated at 37°C for 24 h. Total RNA was extracted using the Trizol reagent (Thermo Scientific) and quantified using the NanoDrop™ One/One C Microvolume UV–Vis spectrophotometer (Thermo Scientific). After reverse-transcription to cDNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific), qRT-PCR was performed using the Quantstudio™ 1 Real-Time PCR System (Thermo Scientific) and Kapa SYBR Fast qPCR kit . PCR was set up as follows: preheating to 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s. β-actin was used as a reference gene. All primer sequences used in this study are listed in Cells were seeded in a 12-well plate at a density of 1 × 105 cells/mL and allowed to grow to complete confluence. The center of resulting monolayers was then vertically scratched using a sterile pipet tip. After a brief washing with PBS, the medium was replaced with serum-free DMEM/F-12, and cells were treated with alpelisib, which was dissolved in 0.1% final concentration of DMSO at various concentrations  and incubated at 37°C for 24 h and 48 h. Migration was examined by microscopy, and pictures were taken. Using ImageJ software , the percentage of wound healing was calculated. Wound closure (%) was calculated based on the following equation: wound (%) = A (t0) − A (t) / A (t0) × 100.To evaluate the migration capacity of CMT cell lines, the scratch assay was used. Cells were seeded in a 12-well plate at a density of 2.5 × 10® Mycoplasma PCR Detection kit, following the manufacturer’s instructions. Briefly, the supernatant from cultured cells was collected, and PCR reaction was performed after mixing it with PCR premix and primer mix. PCR products were then separated using 1.5% agarose gel electrophoresis, and the presence of mycoplasma DNA band was confirmed under UV light using the Invitrogen™ iBright™ CL1500 Imaging System (Thermo Scientific).Mycoplasma DNA was detected using the BioMycoXpost hoc test (Turkey’s multiple comparison against control) to identify statistically significant differences using the GraphPad Prism software. Statistical significance was set as p value < 0.05.All experimental measurements were performed in triplicate, and data are expressed as the mean ± standard deviation (SD). Differences among groups were determined by one-way analysis of variance (ANOVA) with Canine mammary tumor-derived tissues were obtained from the VIP Animal Medical Center and were kindly gifted by the Haedeun Animal Medical Center. Tumors were classified and diagnosed by veterinary pathologists at the veterinary diagnostic laboratories Neodin Vetlab  and IDEXX Laboratories . Histopathological examination of VIP tissue (VT) samples was performed using H&E staining. All VTs were graded based on their morphological characteristics, differentiation, proliferative patterns, and invasive growth into the surrounding tissues. The results of these evaluations are presented in To investigate the frequency of PIK3CA mutation in CMT, gDNA extracted from each VCL and the amplification products were analyzed by Macrogen . Based on previous studies , 24, we We further analyzed the characteristics of VCLs, such as their cell morphology, growth rate, and gene expression, to investigate whether the PIK3CA mutation status is related to the features of canine mammary tumors. Information for each canine patients and the features of the VCL are shown in 50 values in the PIK3CA-mutant cell lines were markedly lower than those in the PIK3CA-wild-type cell lines , 7.203 μM (VCL004), 9.623 μM (VCL005), 4.928 μM (VCL007), and 7.338 μM (VCL009). Meanwhile, the IC50 values of the wild-type cell lines were above 100 μM, except for VCL010 and VCL011, which showed relatively low values. The IC50 value of the normal canine cell line, without to the PIK3CA mutation, was also higher than that of the mutant cell lines.To investigate the effect of alpelisib, a PI3K alpha-specific inhibitor, on the viability of CMT cells, VCLs were treated with various concentrations of alpelisib for 24, 48, and 72 h. In all VCL, the viabilities of cells treated with alpelisib for 24, 48, and 72 h. In all VCLs, the viability of cells treated with alpelisib for 24, 48, and 72 h significantly decreased in a time- and dose-dependent manner . Howeverll lines . PIK3CA-In the normal canine cell line, alpelisib treatment did not affect cell migration, except at the highest concentration for 24 h , and sim50 value in the wild-type VCLs, showed similar mRNA expression levels as the mutant group.To examine whether alpelisib-induced tumor cell death and migration inhibition also affected gene expression, we performed qRT-PCR. The mRNA expression of glucose transporter 1 (GLUT1), also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), which regulates the uptake of glucose in most cell types, was significantly decreased by alpelisib treatment in PIK3CA-mutant cell lines . Even a Mammary cancer is common in both dogs and humans. Canine mammary tumors occur at a high rate in intact female dogs, which is one reason why the majority of female dogs undergoing spaying surgeries. However, according to several studies, the preventive effect on mammary tumors is only observed when spaying is performed at a young age, and spaying female dogs over the age of 2 years does not provide any benefit in preventing mammary cancer , 30. MamMany studies have suggested that PIK3CA is one of the most frequently mutated genes in breast cancer, especially in the hormone receptor-positive (HR+)/HER2- subgroup , 18–20. There are several established canine cell lines derived from mammary cancer, such as CMT-U27, CMT-12, and CMT-7364 –35. HoweAlpelisib is a specific inhibitor designed to target and block the PI3Kα isoform. Alpelisib has been developed and used due to the association between human breast cancer and PIK3CA mutations , 39. HowIn addition to the migration analysis, low-dose alpelisib treatment significantly suppressed cell migration in PIK3CA-mutated cell lines. At the highest concentration of alpelisib, the scratch closure was wider than the initial area owing to toxicity. In contrast, alpelisib did not inhibit the migration of PIK3CA wild-type VCLs. Except for the rapidly growing cells, it can be observed that in the control group, there is no notable difference in the scratch area after 24 h of treatment owing to the low migration rate of these cells. Therefore, it may be challenging to precisely analyze the effects of alpelisib administration. However, when comparing the cell lines that exhibited swift migration ability in the non-treated state, such as VCL002, VCL007, VCL011, and MDCK, the percentage of wound closure was significantly reduced by low-concentration alpelisib treatment in PIK3CA mutants but not in wild-type VCLs. For slow-growing cell lines, the addition of 48 h experiment showed a remarkable difference between PIK3CA mutant VCLs and PIK3CA wild-type VCLs. In the PIK3CA mutant cell line, a high migration rate was observed in the non-treated cells, whereas in the alpelisib-treated cells, migration was significantly suppressed. Meanwhile, the wound closure in wild-type cells and normal canine cells decreased only when treated with a high concentration of alpelisib. These findings suggest that alpelisib effectively suppresses the migration of PIK3CA-mutated tumor cells. However, because the concentration of alpelisib used in the wound healing assay of this study is relatively high, it could potentially affect not only cell migration but also cell death. Therefore, it is necessary to conduct additional experiments using a lower concentration of the drug and transwell system.50 values, which were the lowest observed among the wild-type cells. These results suggested that alpelisib inhibited the glucose metabolism of PIK3CA-mutated cells.Since the PI3K signaling pathway plays a crucial role in energy generation for the survival of cancer cells –43, the Overall, our study revealed that alpelisib is effective against PIK3CA-mutant CMT, similar to its effectiveness against PIK3CA-mutated human breast cancer. Alpelisib may be used more effectively than other drugs in CMT patients when there is a concurrent occurrence of a PIK3CA mutation and a malignant diagnosis, similar to its use to prevent recurrence and metastasis in metastatic human breast cancers with PIK3CA-mutations. Alpelisib could be utilized in the treatment of PIK3CA-mutated benign tumors when surgical excision is not feasible owing to factors such as old age or dog owner’s reluctance. Additionally, we successfully established CMT cell lines characterized by the presence or absence of PIK3CA mutation. Given the similarity between canine and human breast cancers, the cell lines established in this study could be useful for the development of new drugs targeting PIK3CA mutation-derived human breast cancer. However, it is important to acknowledge the limitations of our study, which include a low number of CMT samples analyzed and the lack of investigation into PIK3CA mutation in the normal canines. Further investigations are needed to gain a more comprehensive understanding of CMT. Another limitation is that the growth rate varies among individual cell lines. Because this factor could potentially impact the interpretation of the results, we believe it is necessary to conduct experiments to determine if this changes the outcomes under controlled conditions. Additionally, for cell lines such as VCL008 and VCL012, where chondrocytes might have been mixed with mammary epithelial cells, it would be necessary to further purify the cells through processes such as flow cytometry before they can be used in other studies.In conclusion, this study investigated PIK3CA H1047 and Q546 mutations in CMT and established novel canine cell lines with characteristics PIK3CA mutations. We also demonstrated that alpelisib successfully inhibits tumor cell viability, cell migration, and gene expression related to glucose uptake in canine mammary cells. These findings suggest that the PIK3CA H1047 mutation could serve as a useful prognostic biomarker for CMT, and alpelisib may be a potential drug for targeting PIK3CA-mutant CMT. Further studies are required to investigate the mechanism of action of alpelisib in CMT.https://www.ncbi.nlm.nih.gov/sra/, acession numbers SRR26661417-SRR26661456.The original contributions presented in the study are publicly available. This data can be found here: The animal studies were approved by VIP Research Institute, VIP Advanced R&D: IACUC NON 2022-0002. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.JY: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. YC: Funding acquisition, Project administration, Supervision, Writing – original draft. SA: Funding acquisition, Project administration, Resources, Supervision, Writing – original draft. SJ: Conceptualization, Investigation, Resources, Writing – original draft.