We mainly evaluated whether preserving the inferior mesenteric artery (IMA) sheath to dissecting IMA root lymph nodes  would benefit patients in terms of comparable lymph-node yield removed during operation and postoperative complications in laparoscopic radical resection of rectal cancer.t test, chi-square test or Fisher’s exact test between the 2 groups.This is a prospective study included 141 rectal cancer patients who received laparoscopic radical resection during September 2018 to December 2020. All patients were randomly assigned to the preserved group (n = 71) and the peeled group (n = 70). The baseline characteristics, pathological features, intraoperative and postoperative data outcomes and complications were analyzed by independent samples P = 0.002), a shorter lymph node dissection time (P < 0.001), less intraoperative bleeding (P = 0.004), an earlier time to first flatus (P = 0.013), an earlier time to fluid intake (P = 0.033) and a shorter length of hospitalization (P = 0.012) than the peeled group. The differences between the 2 groups were not statistically significant (P > 0.05) in regard to the total number of lymph nodes cleared, positive lymph nodes, bleeding, anastomotic leakage, pneumonia, wound infection, abscess, ileus, urinary retention, urinary tract infection and chyle leakage.The baseline characteristic and pathological features had no statistical difference between the 2 groups. The preserved group had a shorter operative time   There were 4.064 million new cases of cancer and 2.413 million new deaths occurred in 2016 and showing a continuing upward trend in China [Even so, there are numerous ways to treat rectal cancer, including surgery, radiation, chemotherapy, molecular targeted therapies, immunotherapies, and Chinese herbal medications. Some molecular targeted drugs, such as cetuximab, panitumumab and bevacizumab, have been shown to play important roles in the treatment of rectal cancer –7. Some Different scholars have different opinions regarding laparoscopic radical surgery for rectal cancer, and many studies have been performed on the dissection of No.253 lymph nodes, but few clinical studies have been conducted on the preservation of the IMA sheath during the dissection of No.253 lymph nodes. In the past, we thought that peeling the IMA sheath would increase the detection rate of No.253 lymph nodes in patients with rectal cancer . But nowWe designed this prospective randomized controlled study to investigate whether preserving the IMA sheath is more advantageous in lymph node dissection treatment and postoperative complications compared to peeling the IMA sheath to clean the lymph nodes at the root of the IMA.This is a prospective randomized controlled trial included 141 rectal cancer patients who received laparoscopic radical resection during September 2018 to December 2020, in the Department of Gastrointestinal Surgery, The First Affiliated Hospital of Wannan Medical College. All patients were prospectively and randomly assigned to the preserved group (n = 71) and the peeled group (n = 70) by envelope method. Both groups of patients underwent Dixon surgery for laparoscopic radical resection of rectal cancer, while the patient’s left colic artery (LCA) was preserved during the surgery. Patients with preserved IMA sheaths were placed in the preserved group, and patients with peeled IMA sheaths were placed in the peeled group. All patients and their families communicated with providers and signed informed consent forms before surgery. This study had been performed in accordance with the principles stated in the Declaration of Helsinki. The study was approved by the Ethics Committee of The First Affiliated Hospital of Wannan Medical College and registered by the China Clinical Trials Registry (ChiCTR2200060830).The inclusion criteria were as follows: preoperative pathology showing adenocarcinoma, preoperative diagnosis of stage T1-T3(according to the American Joint Committee on Cancer (AJCC) 7th edition TNM tumor staging criteria) rectal cancer by CT or MRI with a range of 5-15 cm from the anal verge, single rectal tumor lesion with no distant metastases, preoperative American Society of Anesthesiologists (ASA) rating of I-III, no emergency surgery, and no preoperative radiotherapy or other antineoplastic treatment.Exclusion criteria were as follows: conversion to open surgery during laparoscopic surgery; intraoperative finding of mesenteric metastases or local invasion; intraoperative IMA root ligation for bleeding; and patients with comorbid systemic diseases that are inoperable, such as severe coagulation dysfunction, severe liver disease, severe renal disease, severe cardiac disease, severe pulmonary disease or other systemic malignancies. All patients received oral antibiotics 3 days prior to surgery to prevent infection and a cleansing enema 1 day prior to surgery. Those with one or more of the above conditions were excluded. A flow diagram for the study participant screening and grouping is shown in Fig. The researchers, not the operators, put one of the two surgical schemes into an opaque envelope which has the same appearance and size and 100 envelopes are allocated for each scheme. After these envelopes are completely messed up, write a code on the outside of the envelope, seal it and give it to the operator. When a research object enters the study, if it meets the inclusion criteria and exclusion criteria, number the patient, open the corresponding numbered envelope, and perform the surgery according to the grouping scheme in the envelope. The treatment plan received by each subject was determined by the generated random sequence.All the operations were performed by the same team of surgeons following the established grouping requirements. The patient was placed in a supine head-high, leg-low position under general anesthesia via tracheal intubation. Disinfecting the skin, and laying sterile sheets routinely, the abdominal cavity was routinely explored, peeling the right paramedian rectal sulcus with an ultrasound knife. The left mesorectal was separated along the abdominal aorta, the inferior mesenteric plexus was exposed, the IMA was exposed, and lymph node dissection from the beginning of the IMA to the beginning of the LCA without peeling the IMA sheath was performed in the preserved group. In the preserved group, the IMA sheath was not peeled, and the lymph nodes and adipose tissue in the No.253 lymph nodes area were dissected outside the IMA sheath. In the peeled group, the IMA sheath was peeled, and the lymph nodes were dissected at No.253 lymph nodes, the LCA, sigmoid artery (SA) and superior rectal artery (SRA) were exposed. To peel the IMA sheath, we gently peeled the IMA sheath from the root of the IMA with the ultrasound knife and then carefully and gently pushed the nonworking side of the ultrasound knife through the gap between the sheath and the vessel to the bifurcation of the IMA and the LCA, peeling away the intact IMA sheath. Before peeling the IMA sheath, we would carefully free the lymph and adipose tissue at the root of the IMA, expose and protect the root nerve plexus that runs along both sides of the IMA root. Generally, we operated from the point 0.5 cm away from the connection between the IMA and the abdominal aorta to avoid thermal damage to the nerves and blood vessels. The surrounding adipose tissue and lymph nodes were gently dissected, completing the dissection of the No.253 lymph nodes  or Fisher’s exact test. The difference was statistically significant at P < 0.05.All categorical data were measured in numbers or percentages. Microsoft Excel was used for clinical data collection. Statistical Package for Social Science  software was used for statistical analysis of the data. Values were expressed as ‾x ± s, and independent samples A total of 141 patients underwent laparoscopic radical rectal cancer treatment in our hospital between September 1, 2018, and December 31, 2020. They were randomly divided into the preservation group and the peeled group, and the general clinical data of the 2 groups were compared in Table P < 0.001), a shorter operative time , and less intraoperative bleeding  , earlier time to fluid intake (P = 0.033) and shorter hospitalization (P = 0.012). The differences between the 2 groups were not statistically significant  plays an important role in the treatment strategy and prognosis of colorectal cancer, including in peel or laparoscopic surgery or even surgery assisted by the da Vinci robot. Before the appearance of distant metastases, lymph node metastasis is a critical prognostic factor for patients with colorectal cancer and is related to the subsequent treatment plan and course of treatment . ChineseThe lymph nodes at the root of the IMA are classified as third station lymph nodes of the sigmoid colon or rectum. Most scholars think that the area for No.253 lymph nodes dissection is delineated by the IMV on the left, the abdominal aorta on the right, the duodenum on the cephalic side and the area of the angle between the IMA and the LCA on the caudal side , 21. No.The surgical approach of this trial was performed in accordance with the standard laparoscopic radical rectal cancer surgery (Dixon procedure) with preservation of the LCA, while the lymph nodes in No.253 lymph nodes were cleared according to the established requirements for the preserved or peeled groups, and the LCA was preserved because previous research has shown that preserving the LCA in radical rectal cancer surgery can increase the blood supply to the proximal IMA of the colon, provide better conditions for the growth and healing of the anastomosis, reduce the incidence of anastomotic leak and anastomotic stenosis postoperatively and is convenient during laparoscopy –26 .The influence of heteromorphosis of IMA vascular branches on the surgical approach to peeling the sheath should be noted when dissecting No.253 lymph nodes. For IMA vascular typing, Yada  and ShenThe IMA sheath is the tissue that lies between the outer surface of the IMA and the collagen layer that connects the outermost nerve fibers. It contains collagen fibers, nerves, microvasculature, lymphatic vessels and adipose tissue. Studies have shown that microscopically, the IMA sheath contains only lymphatic vessels without the presence of lymph nodes, and no tumor cells are observed within the IMA sheath in lymph node metastasis-positive cases , 31. HowOn the basis of dissection of the IMA sheath. There are currently two types of lymph node dissection for No.253 lymph nodes, and one is to preserve the IMA sheath surgical approach. Li  and GongIn this study, the preserved group had a shorter total operative time than the peeled group. The preservation of the IMA sheath for dissection reduces the operative time, makes the procedure simpler, and reduces the risk of intraoperative anesthetic accidents in patients with a long smoking history, especially those with comorbidities of diabetes, heart disease, kidney or liver problems, which helps to reduce operative risk, improve perioperative safety and improve patient prognosis . The preThe preserved group had significantly shorter times to first flatus and fluid intake than the peeled group. Due to the presence of numerous capillaries and lymphatic vessels within the vascular sheath , 38, we P = 0.735). There was no significant difference in terms of urinary tract infection between the 2 groups. With the use of an ultrasound knife in laparoscopic surgery, the number of patients with chyle leakage in laparoscopic rectal cancer surgery decreased significantly, but there were still cases of celiac disease in the reserved and peeled groups . However, in this study, there was no statistically significant difference between the two datasets.There were no cases of postoperative mortality or deep vein thrombosis after the operation. Although there were cases of postoperative bleeding in the preserved and peeled group, they were all controlled by appropriate treatment. There were four anastomotic leakage patients in the peeled group  and only two in the preserved group . This might be due to the peeling of the sheath affecting the blood flow to the anastomosis, while the influence of the physical condition of the individual patients could not be excluded. Two of the patients with anastomotic leakage were older (both older than 70 years) or had underlying diseases such as hypertension and diabetes. Both groups had three patients with postoperative pneumonia, and all six patients had a long history of smoking. Data from the 2 groups showed no significant difference in the incidences of wound infection and abscess. The number of patients with postoperative ileus is lower than that in the past because surgeons pay more and more attention to the application of enhanced recovery after surgery (ERAS) concept in clinical work. Only two and three patients in both groups developed postoperative ileus; however, the symptoms were quickly alleviated with appropriate and timely treatment. Urinary retention infrequently occurred in both groups, and the data were not significantly different between the 2 groups (5/71, 7.0% vs. 6/70, 8.6% Although this study shows some significant results, there are still some limitations. First, this study is an independent single-center prospective randomized controlled trial, and further validation of the results is needed in a multicenter prospective study with a large sample size. Second, due to limitations in inclusion or exclusion criteria, further data were not available for some patients who had undergone preoperative radiotherapy or emergency cares, and a separate clinical trial could be designed in the future to verify the efficacy of surgery in this population. Finally, in order to reduce the impact of different surgical methods on No.253 of lymph node dissection, the surgical method adopted in this trial was to preserve the LCA for low anterior resection of rectal cancer. Whether the data brought by other surgical methods are similar to this trial needs further prospective trial to verify.Preserving of the IMA sheath in laparoscopic radical surgery for rectal cancer will reduce the total operation time and the length of hospitalization. This surgical method could lead to lower complication rate and faster recovery.


Rutin is a collection of natural compounds that possess a distinctive polyphenolic structure of flavonoids. The present preliminary study aimed to investigate the impact of dietary rutin supplementation on the growth performance, antioxidant capacity, and intestinal health of yellow catfish. Three diets supplemented with different levels of rutin  were fed to juvenile tilapia for 56 days. Results revealed that supplementing with 100 mg/kg rutin enhanced growth, antioxidant capacity, and intestinal health in yellow catfish. However, the administration of rutin at a dosage of 500 mg/kg did not yield any additional advantages but potentially exhibited adverse effects on yellow catfish. This study has demonstrated the potential of rutin as a novel feed additive in aquafeed.Pelteobagrus fulvidraco). Rutin was added to the basal diets at doses of 0 (control), 100 mg/kg, and 500 mg/kg. Each diet was fed randomly into three tanks, each tank containing 30 fish with an initial body mass of (10.27 ± 0.62) g. The feeding trial was conducted in an indoor recirculating aquiculture system at 28 °C for 56 days. According to the findings, the inclusion of 100 mg/kg rutin significantly improved the growth performance of yellow catfish and reduced the feed conversion ratio; however, the growth promotion effect was diminished when the diet was supplemented with 500 mg/kg of rutin. The inclusion of 500 mg/kg rutin in the diet significantly reduced the level of crude lipid and protein of the whole fish. Serum activities of alkaline phosphatase, albumin, and total protein were all significantly increased when fish were fed the diet supplemented with 500 mg/kg rutin, while serum glucose was significantly lower compared to the control group. Meanwhile, dietary rutin at a concentration of 500 mg/kg significantly induced the hepatic mRNA expressions of antioxidant-related genes  and inflammatory-associated genes . Incorporating rutin at doses of 100 mg/kg and 500 mg/kg into the diets resulted in a notable increase in superoxide dismutase (SOD) activity, while simultaneously reducing malondiadehyde (MDA) content in the liver and intestine. Intestinal villus height, villus width, muscular thickness, and lumen diameter were significantly increased with the administration of 500 mg/kg of dietary rutin. Gut microbial diversity analysis indicated that supplementing diets with 100 mg/kg and 500 mg/kg rutin significantly enhanced the abundance of Cetobacterium while decreasing Plesiomonas richness. In conclusion, dietary rutin levels at 100 mg/kg could enhance the growth, antioxidant capability, and intestinal health of yellow catfish under present experimental conditions.This research aimed to examine the effects of dietary rutin supplementation on growth, body composition, serum biochemical indexes, liver enzyme activities and antioxidant-related genes expression, intestinal morphology, and microbiota composition of juvenile yellow catfish ( Ctenopharyngodon idella) [Rhamdia quelen) resulted in increased antioxidant enzyme activities in the brain, liver, kidney, and gills while also promoting growth [Oreochromis niloticus) [Rutin is a collection of natural compounds that possess a distinctive polyphenolic structure of flavonoids . It exhi idella) . The admg growth . Furtherloticus) .Pelteobagrus fulvidraco) is a widely cultured fish species in China. Its production reached about 0.58 million tons, which ranked tenth in total freshwater fish output [Yellow catfish . Based on the previous literature ,12, dietn = 30) were chosen and randomly divided into 9 tanks . Three tanks were randomly divided into groups and fed one of the test diets. The fish were fed three times a day , and the feeding rate was 3–5% of body weight. The fish were weighed once every two weeks, and the feeding rate was adjusted according to the change of body weight. The feeding trial went on for 56 days. Feed intake and mortality of test fish were recorded daily. The filter sand tank of the RAS was backwashed with about 10% fresh water before feeding every morning and afternoon. The water temperature was kept at 28 °C. During the culture experiment, water quality parameters were measured once a week. The main water quality parameters were dissolved oxygen > 5 mg/L, pH: 6.5–7.5, total ammonia < 0.2 mg/L, nitrite < 0.05 mg/L.The experimental yellow catfish were provided by a local fish breeding facility. The experimental fish were acclimated for 4 weeks in an indoor recirculating aquiculture system (RAS) at Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. During the domestication period, the fish were hand-fed three times a day with apparent satiation. Prior to the official feeding trial, the fish were fasted for 24 h. Afterwards, 270 yellow catfish with initial weight , hepatosomatic index (HSI), and condition factor (CF). Part of the liver and midgut of 3 fish per tank were placed in frozen tubes, frozen in liquid nitrogen, and stored in the refrigerator at −80 °C for the determination of liver metabolomics, antioxidant indexes, and intestinal microorganisms. An additional portion of the liver and the midgut of 6 fish per tank was used for the preparation of HE tissues sections for histological observation.At the end of the feeding trial, the fish were fasted for 24 h, then counted and weighed in buckets, and the weight gain rate and specific growth rate were calculated. Six fish per tank were anesthetized with 75 mg/L MS-222, and their body length and weight were measured. Blood was drawn through the tail artery and allowed to stand for 4 h, then centrifuged at 960× Approximate analyses of whole fish and experimental diets were performed according to the method of the Association of Official Agricultural Chemists . In briThe contents of serum total protein (TP) , albumin (ALB) , triglyceride (TG) , total cholesterol (TCHO) , glucose (GLU) , and the activities of alkaline phosphatase (ALP) , aspartate aminotransferase (AST)  and alanine aminotransferase (ALT)  were detected by an automatic biochemical analyzer . Diagnostic reagents were obtained from Sysmex Wuxi Co., Ltd.  according to standard protocols.w/v) of ice-cold saline solution; it was centrifuged at 960× g for 10 min at 4 °C to separate the supernatant. Superoxide dismutase  and malondialdehyde  were measured by biochemical kits .The tissue was thoroughly homogenized with 10 volumes . Five fields were randomly selected from each intestinal section to measure the number, height, width, diameter of mucosal fold, and muscular layer thickness using Image pro plus 6.0 software .The midgut samples from the R0, R100, and R500 groups were collected for DNA extraction. After the measurement of DNA integrity and purity, about 2 ng/μL DNA for each sample was used for 16S rRNA sequencing. The primer sets including 338F (5′-ACTCCTACGGGAGGCAGCA-3′)/806R (5′-GGACTACHVGGGTWTCTAAT-3′) were utilized to amplify variable V3–V4 regions of the 16s rRNA. The sequencing was accomplished by the staff of Shanghai Meiji Biotechnology Co., Ltd.  with the Illumina Miseq platform. Bioinformatics analysis was described in detail following our recently published literature . RepreseTotal RNA was extracted from liver samples of groups R0, R100, and R500 using Trizol reagent , according to the manufacturer’s instructions. RNA integrity was verified by 2% agarose gel electrophoresis, and the concentration and purity were determined using a Nanodrop spectrophotometer . Two μg of RNA from each sample was reverse transcribed to first-strand cDNA using the primer-scriptTM RT kit (Takara Biotechnology).Cu/Zn-SOD), superoxide dismutase 2 (Mn-SOD), catalase (CAT), glutaredoxin 3 (GPx), alpha-induced protein 8-like 1 (TNFα), IL-10 (interleukin 10), lysozyme g-like (LYZ), and ribosomal protein L13a (Rpl13a) were selected as target genes for detection. These primer sequences are listed in β-actin was used for gene normalization. Relative mRNA expression levels were calculated according to the 2−ΔΔCt method [qRT-PCR was performed using an ABI 7500 Real-Time PCR system with a reaction volume of 20 µL, 10 µL SYBR Premix Ex Taq II (Takara Biotechnology), 8.2 µL double-distilled water, 1 µL cDNA template, and 0.4µL primer (10 µM) included. All reactions were performed three times. Superoxide dismutase 1 (t method .n = 3), and p < 0.05 indicated significant difference.SPSS20.0  was used for one-way analysis of variance (one-way ANOVA) of the experimental data, and Duncan’s multiple comparison analysis was used to analyze the significance of the difference between groups. All data results were expressed as mean ± standard deviation  than the fish fed diet R0. The fish fed with diet R100 exhibited a significantly higher weight gain rate and specific growth rate compared to the fish fed with diet R0 (p < 0.05), and there was no significant difference between group R500 and group R100 (p > 0.05). There were no significant differences on the hepatosomatic index, condition factor, and the viscerosomatic index of yellow catfish among the groups (p > 0.05).p < 0.05). The crude lipid content of the whole fish in the R500 group was significantly lower than that of the other two groups (p < 0.05). Dietary rutin supplementation did not affect the moisture and ash contents of the whole body (p > 0.05).The proximate compositions of whole fish are presented in p < 0.05). Fish in the R100 and R500 groups had notably reduced levels of glucose (GLU) and activities of alanine transaminase (ALT) compared to the fish in the control group (p < 0.05). Nonetheless, the experimental groups did not display any noteworthy variations in serum total protein (TP), triglyceride (TG), total cholesterol (TCHO), and aspartate aminotransferase (AST) activity (p > 0.05).p < 0.05), and the MDA content was significantly lower in the R100 and R500 groups compared to the R0 group (p < 0.05).The superoxide dismutase (SOD) activity and malondiadehyde (MDA) content of yellow catfish are displayed in Cu/Zn-SOD, Mn-SOD, CAT, and GPx) and inflammation-related genes  in the R500 group were significantly up-regulated among the three groups. The R100 group had the lowest transcriptional expression of Mn-SOD, CAT, and GPx among the three groups. However, there was no significant difference in the expression of inflammation-related genes between the R100 group and the control group.The results of genes expression analysis are shown in p < 0.05).As shown in p > 0.05). The Chao and Ace index in the R500 group was significantly higher than that in R0 group (p < 0.05). At the phylum level, Fusobacteria, Proteobacteria, and Firmicutes were the dominant phylum in the gut flora of yellow catfish . In addition, the change in blood glucose is correlated with hepatic enzymes activities, which may suggest that rutin has a beneficial influence on liver function. Notably, reduced AST activity observed in fish fed with rutin further supports its potential for improving overall liver function when compared to the control diet.Serum biochemical parameters generally reflect the fish physiological and metabolic conditions under nutritional manipulation or environmental stress . TP coulH&E staining is commonly used for histopathology evaluation in fish due to that it can clearly illustrate cellular structures with remarkable signatures . ExcessiCetobacterium, Plesiomonas, and Peptostreptococcaceae were identified as dominant genera within the gut microbiota of yellow catfish. Cetobacterium can produce vitamin B12 in the process of carbohydrates fermentation and play crucial functions in the nutritional metabolism [Peptostreptococcaceae predominated in gut diseases [Cetobacterium and the decreased Plesiomonas richness were determined on fish fed the diets with 100 mg/kg or 500 mg/kg rutin. These data suggested that dietary rutin has positive effects on the improvement of intestinal structure and microbial composition, which is beneficial to the gut health of yellow catfish.There exists a substantial population of microorganisms within the fish intestine which has established a dynamic and intricate microenvironment over an extended period of evolution . The guttabolism . Pseudomdiseases . The preIn summary, the present results demonstrated that dietary supplementation of rutin at 100 mg/kg could improve the growth, antioxidant capability, and intestinal health of yellow catfish. However, the administration of rutin at a dosage of 500 mg/kg did not yield any additional advantages but potentially exhibited adverse effects on yellow catfish. The determination of the optimal quantity of rutin to be included in the diet of yellow catfish will be conducted in forthcoming investigations.


Psoriasis is an immune‐mediated chronic inflammatory disease, and currently it is widely believed that the IL‐23/IL‐17 axis and Th17 cells play a critical and central role. However, increasing evidence suggests that neutrophils may interact with a variety of immune cells to play an indispensable role in psoriasis.We searched the recent literature on psoriasis and neutrophils through databases such as PubMed and CNKI, and summarized the findings to draw conclusions.Neutrophils can promote the development of psoriasis by secreting IL‐23, IL‐17, and cytokines with TH17 cell chemotaxis. Activated keratinocytes (KCs) can attract and activate neutrophils, induce the formation of neutrophil extracellular traps (NETs). KCs can also expose self‐antigens which lead to strong autoimmune reactions. The granule proteins secreted by activated neutrophils can activate IL‐36, which converts vulgaris psoriasis to generalized pustular psoriasis (GPP).The function of neutrophils components and the interaction between neutrophils and immune cells play an essential role in the pathogenesis of psoriasis. The aim is to provide a theoretical basis for the exploration of targeted clinical treatments and fundamental research on the pathogenesis of psoriasis. They play an indispensable role in anti‐infective and inflammatory processes through mechanisms such as antigen presentation, reactive oxygen species (ROS) generation, release of granular proteins, formation of neutrophil extracellular traps (NETs), and production and release of cytokines.1.1Neutrophils, as a type of innate immune cell, are the first to reach the site of inflammation in the early stages of inflammation. Neutrophil lifespan is short, and apoptosis begins after entering the blood for 24 h, but during the period of psoriasis inflammation, the lifespan of neutrophils can be significantly extended.1.1.1IL‐23, composed of subunits p40 and p19. Macrophages, dendritic cells, and KCs in psoriatic skin tissue can secrete IL‐23After binding to IL‐23R, IL‐23 can exert various biological effects. IL‐23R is expressed on the surfaces of lymphocytes and myeloid cells .1.1.2It is currently believed that IL‐17 plays an important role in psoriasis, and IL‐17 antagonists such as secukinumab have been widely used in clinical practice.Among other diseases comorbid with psoriasis, IL‐17‐producing neutrophils have also been observed. IL‐17+ neutrophils were found in atherosclerotic plaques.1.2Neutrophils can be recruited by various chemokines and migrate to the lesion site. KCs can produce neutrophil chemokines, but previous studies have shown that neutrophils themselves can also produce chemokines under the stimulation of inflammation,1.3MPO gene defects have decreased ability to produce NETs.The composition of neutrophil granule proteins is relatively complex and can be divided into three major categories, including primary azurophilic granules (myeloperoxidase (MPO) etc.), secondary specific granules, and gelatinase granules  etc.).1.41.4.1NETs are fibrous networks released by neutrophils that contain various components such as neutrophil granule proteins, LL‐37, RNA, and cytokines.The formation of extracellular traps by neutrophils is called NETosis.The uncontrolled release of NETs may contribute to various diseases such as psoriasis, systemic lupus erythematosus,1.4.2NETs contain MMP‐9,Both mast cells and neutrophils release IL‐17 during the formation of extracellular traps, but neutrophil NETs contain higher levels of IL‐17 than MCETs do.1.5In traumatized or microbially infected skin, mast cells recognize viruses, bacteria, and other pathogens through TLRs.KC in the skin has antigen‐presenting function.When activated by pathogenic factors in the internal and external environment, KCs release neutrophil‐derived chemokines, which results in the recruitment of neutrophils to the skin. When neutrophils arrive at the vessels in the skin, they interact with the VECs. The MMP‐9 produced by neutrophils destroys cell connections between VECs, causing vascular dilatation and increased permeability. Along with changes on vascular endothelial cells, neutrophils are activated,2Neutrophils are innate immune cells that play a critical role in the early stages of psoriasis inflammation. They can interact with other immune cells such as KCs and mast cells through the production of IL‐17, formation of NETs, and release of granule proteins. Neutrophil lifespan is significantly extended, and KCs and TH17 cells can attract neutrophils, amplifying the inflammatory effect.Previous studies have identified the role of NETs in psoriasis, atherosclerosis, uremia, inflammatory bowel disease, and chronic obstructive pulmonary disease. NETs also play an important role in the pathogenesis of comorbidities. Today several drugs that can inhibit NETs have been developed for possible blocking NETosis, including cannabidiol [99],The authors have no relevant financial or nonfinancial interests to disclose.


Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56αCme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the β-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment. Loss of PP2A activity is often associated with cancer but the underlying mechanism remains unclear. Here, the authors show that decreased methylation of PP2A catalytic C subunit caused by loss of LCMT-1 in prostate cancer abrogates the tumor suppressor activity of PP2A on AR/MED1-dependent gene expression, proposing decreased methyl-PP2A-C as a prognostic marker for prostate cancer progression. Though the inappropriate activation of numerous oncogenic kinases plays a causal role in transformation, metastasis, and therapy resistance and several of these kinases have been successfully targeted for cancer treatment, much less is known about the function and mechanisms of regulation of phosphatases in cancer. The highly conserved protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase with a complex heterotrimeric composition involved in the regulation of numerous critical aspects of cellular function2. PP2A is one of the most abundant enzymes, accounting for up to 1% of the total cellular protein in some tissues and represents the majority of serine/threonine phosphatase activities in many tissues3. PP2A is comprised of a core dimer consisting of a scaffolding “A” subunit (PPP2R1/PP2A-A), and a catalytic “C” subunit (PPP2C/PP2A-C), which form a heterotrimeric complex with one of at least 20 substrate-directing regulatory “B” subunits clustered into four structurally distinct families PPP2R5/B56, PPP2R2/B55, PPP2R3/PR72/PR130, and Striatin4. The AC heterodimers are not considered the functional phosphatase holoenzyme in vivo, with the binding of the B subunit conferring target specificity and allowing PP2A to dephosphorylate target proteins.Reversible protein phosphorylation plays a fundamental role in numerous biological processes and is regulated by a dynamic interplay between protein kinases and phosphatases. Disruption of this balance due to aberrant activation of kinases and inactivation of phosphatases is a hallmark of cancer6. PP2A dysregulation is commonly observed in cancer, typically through one of several mechanisms, including somatic mutations, haploinsufficiency and/or decreased expression of PP2A subunits, and increased expression of endogenous PP2A inhibitors such as CIP2A10. PP2A heterotrimerization is regulated through post-translational modifications of the catalytic C subunit, which influence the binding affinity of the B subunits to the AC dimer and thus the substrate specificity. While PR130 and Striatin binds to the AC dimer in a methylation-independent manner, a central evolutionarily conserved mechanism that leads to heterotrimerization of AC and B55, and B56 subunits is the S-adenosylmethionine dependent PP2A-C α-carboxymethylation at the terminal leucine residue (L309) by Leucine Carboxyl Methyltransferase 1 (LCMT1)14. The presence of methylation on L309 neutralizes the negative charge on the terminal carboxylic acid, allowing for the binding of both B55 and B56 family members to the PP2A AC dimer. Conversely, Protein Phosphatase Methylesterase 1 (PME-1) mediated removal of this methylester from the PP2A-C dimer results in the retention of the negatively charged terminal carboxylic acid creating steric hinderance that prevent B55 and B56 family member binding, thus adding another layer to the regulation of PP2A biogenesis16. Given the previous studies demonstrating the role of the B55/B56 methyl-sensitive PP2A heterotrimers in regulating gene expression by targeting multiple signaling pathways such as PI3K/AKT/mTOR activation20, one critical function of C subunit methylation by LCMT1 could be to coordinately regulate both B55/B56 containing heterotrimers to negatively affect transcription and survival signals in cancer. Interestingly, LCMT1 is the only enzyme known to catalyze methylation of not only PP2A-C but also two other PPP family members, the PP4 and PP6 catalytic subunits22. Nevertheless, the status of LCMT1 expression, regulation, and its role in cancer development and progression remains elusive.PP2A is widely considered a tumor suppressor and plays a fundamental role in regulating cellular homeostasis by dephosphorylating a broad array of protein targets, from mitotic spindle components, chromatin regulators to intracellular signaling mediators23. PCa relies on AR driven transcription for growth and survival, forming the basis for effective androgen deprivation therapy (ADT) as a first-line of treatment in the primary stages of the disease. However, restoration of AR signaling through AR point mutations, AR overexpression, AR genomic amplification, and constitutively active AR splice variants contributes to an AR-addicted castration-resistant prostate cancer (CRPC)26. Next-generation AR signaling inhibitors (ARSIs) such as abiraterone, enzalutamide, darolutamide, or apalutamide are used in combination with ADT to treat metastatic and nonmetastatic CRPC28. Despite the clinical success surrounding ARSI, resistance to these therapies invariably emerges through various mechanisms that restore AR-signaling, resulting in poor clinical outcomes31. AR-mediated transcriptional addiction is, therefore, a hallmark of a vast majority of refractory CRPC, where AR functions in a multi-protein complex comprising transcriptional coactivators and chromatin-associated proteins33. Additionally, greater than 50% of CRPC demonstrates PI3K pathway activation through either biallelic loss of Pten or activating mutations in PIK3CA and AKT134. Nevertheless, how this recurrently activated oncogenic survival pathway converges upon AR signaling is unclear. There remains an impending need for a better understanding of the mechanism that restores AR signaling and to discover novel ways to target AR addiction in refractory CRPC.Prostate cancer (PCa) is the second most common cancer in men worldwide, and the eighth leading cause of cancer-related deathsIn this study, we report the association of decreased PP2A-C α-carboxymethylation of L309 with rapid biochemical recurrence and metastasis in PCa. Our results show that reduced AB56αC heterotrimers lead to increased AR activity and castration-resistant tumor growth in vivo. Furthermore, we demonstrate LCMT1-dependent methyl-sensitive AB56αC heterotrimer targets AR and its critical coactivator MED1 for dephosphorylation, resulting in the decruitment of the AR-MED1 complex from the chromatin and loss of target gene expression. In addition, we identified S6K1/β-TRCP mediated degradation of LCMT1 as a mechanism for the restoration of AR signaling in antiandrogen refractory prostate cancer cells. Together, these results demonstrate that the loss of LCMT1/decreased PP2A-C α-carboxymethylation is a major determinant of AR-addicted PCa and supports the therapeutic use of AR degraders and selective small molecule modulators of PP2A for refractory prostate cancer treatment.22. To better define the role of carboxymethylation in regulating B56 holoenzyme formation, we specifically immunoprecipitated B56α . This was different for the B56α subunit, which showed similar total expression levels in wild-type and Lcmt1 null cells. However, B56α had a reduced ability to interact with non-methylated PP2A-C/PP2A-A dimers indicating a high methyl-PP2A-C affinity/preference of B56α. Re-expression of V5-tagged LCMT1 in Lcmt1 null cells resulted in a quantitative rescue of the defects in PP2A-C methylation as well as in B56 holoenzyme assembly  with antibodies against p-MED1 and p-AR revealed nuclear puncta for both factors in LNCaP cells expressing LacZ, however, a significant reduction in the puncta and total fluorescence accompanied by increased methyl-PP2A-C staining was observed in cells overexpressing LCMT1  and four (isoleucine) to alanine (2A)  using CRISPR. Ectopic expression of HA-tagged MED1 and Halo-tagged AR showed increased p-MED1 and p-AR in the LCMT1 null cells compared to Cas9 control, and interestingly, the phosphorylation of endogenous MED1 was substantially higher in the null cells, suggesting a direct role of LCMT1 via PP2A in regulating MED1 and AR phosphorylation Fig. . Next, iMT1 Fig. . Next, cMT1 Fig. . Since P AR Fig. . The two2A) Fig. . Co-tran2A) Fig. . These o2A) Fig. , which e2A) Fig. . Collect2A) Fig. , thereby2A) Fig. . We haveant Fig. , thus suant Fig. .Fig. 3LCklk3 and others  or cullin neddylation inhibitor (MLN4924) led to the accumulation of LCMT1, implying a potential cullin-RING E3 ubiquitin ligases (CRLs) dependent mechanism in regulating LCMT1 stability and degradation amino]propan-2-olato dizinc(II) specifically binds to phosphorylated amino acids and generates a mobility shift on acrylamide gels proportional to incorporated phosphates48, we observed a slow-migrating band(s) for LCMT1, which was eliminated upon phosphatase ) treatment  such as phosphorylation50. Noticeably, we identified a conserved phosphorylation consensus R/X/R/X/X/S/T sequence (R4QRESS9), for the protein kinase AKT-mTOR-ribosomal protein S6 kinase 1 (S6K1) in its N-terminal IDR  Pten null prostates develop high-grade prostate intraepithelial neoplasia (HGPIN) phenotype- the main precursor lesion to adenocarcinoma53, and they displayed a marked increase in p-MED1 staining, along with higher MYC, p-AKT and p-S6 staining - a characteristic pattern associated with LCMT1 loss/reduction  used in the study showed no significant pathological changes. Similar to observations made in human cells, PI3K-AKT-mTOR pathway inhibitors stabilized LCMT1 in mouse fibroblast cells , apalutamide (Apa), and darolutamide (Daro) are efficacious in the treatment of both nonmetastatic and metastatic CRPC27. However, the disease invariably recurs through restoration of AR-signaling55. Our observation that decreased LCMT1 expression leads to increased p-AR/p-MED1, reduced sensitivity to enzalutamide, and castration-independent growth  in LNCaP and VCaP cells by growing them continuously in the presence of the drug for close to six months and collecting the cell fractions at regular intervals. As expected, following a quiescence associated reduction in p-AR/AR and p-MED1/MED1 for the first 6-7 weeks, a steady increase in their level was evident, paralleled by a gradual loss in LCMT1 and methyl-PP2A-C, as cells evolved to attain the EnzaR state  engineered from phenothiazine parent compounds have shown to drive dephosphorylation of select pathogenic substrates, including AKT and S6K162. From our finding implicating S6K1 in directly regulating LCMT1 expression resulting in stable AR transcriptional complex formation in refractory CRPC cells, we postulated that inhibition of AKT-S6K1 signaling by these PP2A modulator molecules will have a feedforward effect on LCMT1-dependent AB56αC heterotrimer assembly and AR/MED1 dephosphorylation , the human genome codes for far fewer (∼50) putative serine/threonine phosphatases (PSPs) comprising three major families: phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases (PPMs), and the aspartate-based phosphatases represented by FCP/SCP 38. Our data show a reduction in the AB56αC/methylation-sensitive heterotrimers due to phosphorylation-mediated degradation of LCMT1 involving S6K1—a downstream effector of the PI3K pathway. This strongly suggests a paradigm where persistent oncogenic PI3K signaling in cancer cells is achieved by the elimination of its negative regulator AB56αC and also other methylation-sensitive heterotrimers such as AB55C through S6K1-mediated degradation of LCMT1. Another important implication of these findings is that the PI3K signaling could also result in the inactivation of other phosphatase family members, such as PP4 and PP6, since LCMT1 is also required for the methylation of PP4-C and PP6-C catalytic subunits21.Moreover, AB56αC/methylation-sensitive heterotrimers negatively regulate the PI3K-AKT-mTOR-S6K signaling axis, and a high proportion of prostate cancers exhibit increased activation of this critical survival pathway73. However, though the dogma of PP2A-regulated transcription is well established, we here report the presence of canonical PP2A heterotrimer components on the chromatin. In this regard, our mechanistic data show LCMT1 catalyzed methylation-sensitive AB56αC heterotrimers on the chromatin with consequent dephosphorylation of AR-MED1, thus providing the evidence for a direct PP2A-substrate interaction on the chromatin and a consequent change in gene expression. Recently, a noncanonical PP2A holoenzyme, comprising the AC dimer and the multi-subunit RNA endonuclease integrator, Integrator–PP2A complex (INTAC), was shown to regulate and fine-tune transcription and RNA maturation by antagonizing the action of the CDK9 kinase activity on targets like DSIF and RNAPII74. However, it is unclear whether the interaction between the AC dimer and Integrator complex is methylation dependent; if so, then LCMT1 could be expected to facilitate not only the canonical substrate-specific PP2A heterotrimers but also Integrator-mediated transcriptional control directly through L309 methylation of PP2A-C.Methylation-sensitive PP2A heterotrimers have also been implicated in the regulation of oncogenic transcription through negative regulation of chromatin modulators and transcription factors such as BRD4, HDACs, SWI/SNF, and MYC30. Though restoration of AR signaling through AR point mutations, overexpression, genomic amplification, and constitutively active splice variants is shown to promote resistance to AR antagonism, the underlying molecular basis for AR transcriptional activity and its requirement for the survival of ARSI-resistant cells is unclear56. The findings here implicate AKT/S6K1-mediated LCMT1 degradation as key to developing resistance to ARSI in CRPC cells that continue to rely on AR activity for survival. Furthermore, our data showing the sensitivity to AR degraders in the ARSI-resistant cells illustrate the incomplete targeting of AR by enzalutamide, apalutamide, or darolutamide. Therefore, further targeting of the AR through PROTAC-based therapies that are under investigation75, may be clinically beneficial in patients who develop resistance to next-generation ARSI. Furthermore, the loss of methylation-sensitive AB56αC heterotrimers, and potentially AB55C heterotrimers which is known to negatively regulate AKT76, leads to increased PI3K/AKT signaling in ARSI refractory CRPC cells. This suggests that drugs targeting PI3K/AKT could be beneficial in treating ARSI refractory CRPC.Despite recent advances in treating CRPC with second-generation ARSI such as enzalutamide, darolutamide, and apalutamide, de novo resistance is observed in 20–40% of patients, and most patients invariably acquire resistance to these therapies77 based therapeutics could provide additional opportunity for targeted activation of methyl-sensitive biased PP2A heterotrimers in treating transcription addicted cancers.Beyond their biological significance, the results presented in this work strongly support the use of SMAPs in treating ARSI-resistant CRPC. The SMAP induced biased PP2A reactivation coupled with feedforward activation of LCMT1 through AKT/S6K1 inhibition provide an excellent opportunity for targeted eviction of the hyperphosphorylated AR transcriptional complex from chromatin. While target-specific molecular glues like SMAPs are still in their early phase of development, our study suggests that combining PI3K/AKT/S6K1 pathway inhibitors with antiandrogen therapies cannot be ruled out in AR-addicted prostate cancer. Furthermore, the mechanistic insights presented in this work indicate that direct stabilization of LCMT1 by phosphorylation-targeting chimeras (PhosTACs)Pten knockout mice were generated by crossing Pbsn-Cre male mice Tg (Pbsn-cre) 4Prb/J  with Pten floxed mice . All mice were housed in a pathogen-free animal barrier facility and all in vivo experiments were initiated with male mice aged 5-8 weeks.Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Pennsylvania and/or The University of Michigan. Animal use and care was in strict compliance with institutional guidelines and all experiments conformed to the relevant regulatory standards by the Universities. The maximal tumor size in our study models (mice) never exceeded 1 cm in diameter as allowed by the above ethics committee. NOD SCID or NCI SCID/NCr athymic nude mice were obtained from the Jackson Laboratory (strain code: 005557) and Charles River (strain code: 561). Prostate-specific The maintenance of mice and the experimental procedures (immunizations) have been conducted according to the Austrian Animal Experiments Act and have been approved by the Austrian Federal Ministry of Science and Research BMWFW-66.009/0211-WF/V/3b/2015 and the animal experiments ethics committee of the Medical University of Vienna.78. LNCaP, LNCaP-AR, VCaP, and LAPC4 enzalutamide-resistant cell lines were generated by culturing their parental lines in presence of enzalutamide (10 µM) for 4–6 months in vitro. Surviving enzalutamide-resistant polyclonal pools were maintained in 10 µM enzalutamide throughout. Apalutamide- and darolutamide-resistant LNCaP, LNCaP-AR, VCaP, and LAPC4 lines were generated by culturing the cells in 10 µM of the respective drugs for at least two months.The cell lines used in this study were obtained from American type cell culture (ATCC) unless mentioned. LNCaP-AR cells were a generous gift from Dr. Charles Sawyers- Memorial Sloan-Kettering Cancer Center, New York, NY. LNCaP, LNCaP-AR, and LAPC4 prostate cancer cell lines were grown in RPMI 1640 , VCaP- prostate cancer line, HEK293-T, and NIH-3T3, cells were grown in DMEM with Glutamax . HAP1 wild-type and LCMT1 CRISPR Cas9 deletion cells  were grown in Iscove’s Modified Dulbecco’s Medium . The medium was supplemented with 10% of FBS  or FCS  and 1% of Penicillin Streptomycin Solution . LNCaP-AR enzalutamide-resistant cell lines were derived from enzalutamide-resistant tumor xenografts as described previously2. The complete list of cell lines used is indicated in Supplementary Data All experiments using antiandrogen-resistant pools were done in the presence of enzalutamide, except when cells were treated with AR-PROTAC ARD-69. shLCMT1 or non-targeting control shNTC cell lines were created transducing shLCMT1-GFP or shNTC-GFP lentiviral vectors in LNCaP, VCaP, LAPC4, and LNCaP-AR cells. Cells were sorted for GFP expression. Knockdown was confirmed by immunoblotting. LCMT1 overexpression or control cell lines were generated by transducing pLX304-LacZ or pLX304-LCMT1 in LNCaP cells. Cells were selected using Blasticidin. Overexpression was confirmed by immunoblot. All the cell lines were routinely tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit  and were maintained in a humidified incubator at 37 °C and 5% COThe complete list of the reagents used throughout the study and their commercial suppliers are given in Supplementary Data 79.A set of tissue microarray (TMA) slides (section thickness 3 µm) containing a single 0.6 mm tissue punch from each of 17,747 patients was constructed at the Institute of Pathology, University Medical Center Hamburg-Eppendorf, Germany. The composition of the TMA is given in Supplementary Table 2O2 and protein exposure to remove non-specific binding. The slides were exposed overnight to the antibody . After washing with TBST. The slides were exposed to DAKO Envision+ (Goat antimouse polymerized HRP) and subsequently the chromogen, diaminobenzidine (DAB). After counterstaining with hematoxylin, the slides were digitally scanned using an APERIO scanner. In order to determine the specificity of the antibody, the antibody was pre-incubated with 10x molar excess of the methylated and unmodified peptides prior to incubation with slides.Immunohistochemistry was performed on a DAKO autostainer. TMA slides were deparaffinized and rehydrated in a graduated series of ethanol. Slides were subjected to epitope retrieval using 10 mM Tris–HCl buffer pH9.0 containing 1 mM EDTA. The slides were then subjected to HThe digital slides were examined using QuPath and the H-score and Allred scores were submitted back to the originator of the TMA slides to be correlated with the clinical data.2O2 in a sodium acetate buffer pH = 6. The reaction was stopped by the addition of 1 N H2SO4 and the absorbance was measured at 450 nm, for background correction the absorption of 560 nm was subtracted. For ELISA quantification the signals of the antibodies were normalized to the signal of the cognate target peptide of the antibody which was artificially set to 1.ELISA 96-well plates  were coated with 50 µl peptide solution (2 µg/ml in TBS) at 4 °C over night. The plate was blocked with 2% BSA in TBS for 1 h at RT and incubated with primary antibodies in TBS for 1 to 2 h at room temperature. Incubation with secondary peroxidase conjugated antimouse was performed for 1 h at RT followed by detection with TMB  and HMeasurements were performed using Biacore T200 instrument. The sensorgrams obtained were analyzed with Biacore T200 Evaluation Software (version 3.1). Flow cells were coated with 30 µg/mL antimouse IgG antibody (in immobilization buffer) using the mouse antibody capture kit  via amine coupling according to the manufacturer’s protocol. The immobilization levels obtained were 11605.9 and 11333.9 RU. The antibody (7C10) was applied with a concentration of 50 µg/ml and reached a response level of approx. 1200 RU. The antigens were used in different concentrations:PP2A Me 5 nM, 10 nM, 20 nM, 40 nM, 80 nM and 160 nM for 7C10PP4 Me 50 nM, 100 nM, 200 nM, 400 nM, 800 nM for 7C10Antibody capturing time was adjusted to reach approx. 1200 RU, antigen association time was 360 s, dissociation time was 450 s. The flow rate was 40 µl/min. Regeneration of the chip was performed injecting 10 mM glycine-HCl pH 1.7 for 180 s with a flow rate of 20 µl/min. The value of the equilibrium dissociation constant (KD) was obtained by fitting a plot of response at equilibrium (Req) against the respective concentration of the analyte .Peptides were purchased from Pichem. The sequences of the peptides used in this study are as follows:309: Ac-His-Val-Thr-Arg-Arg-Thr-Pro-Asp-Tyr-Phe-Leu-OHLeu309: Ac-His-Val-Thr-Arg-Arg-Thr-Pro-Asp-Tyr-Phe-Leu-OMemeLeu309: Ac-His-Val-Thr-Arg-Arg-Thr-Pro-Asp-Tyr-Phe-Leu-NH2amLeu307: Ac-Ile-Pro-Ser-Lys-Lys-Pro-Val-Ala-Asp-Tyr-Phe-Leu-OHLeu307: Ac-Ile-Pro-Ser-Lys-Lys-Pro-Val-Ala-Asp-Tyr-Phe-Leu-OMemeLeu307: Ac-Ile-Pro-Ser-Lys-Lys-Pro-Val-Ala-Asp-Tyr-Phe-Leu-NH2amLeu305: Ac-Ile-Pro-Pro-Arg-Thr-Thr-Thr-Pro-Tyr-Phe-Leu-OHLeu305: Ac-Ile-Pro-Pro-Arg-Thr-Thr-Thr-Pro-Tyr-Phe-Leu-OMemeLeu305: Ac-Ile-Pro-Pro-Arg-Thr-Thr-Thr-Pro-Tyr-Phe-Leu-NH2amLeuFor immunoblotting (IB), cells were harvested and lysed by sonication in RIPA buffer - , supplemented with protease inhibitor cocktail  and phosphatase inhibitor . Protein concentration was determined by the standard BCA method, and an equal amount of protein from each sample was boiled in a sample buffer and subjected to SDS–PAGE. The proteins were transferred to PVDF membrane  or nitrocellulose membranes, blocked with PBS-T - 5% (w/v) nonfat milk in PBS containing 0.1% Tween-20, probed with the relevant antibodies for 2 h at room temperature or overnight at 4 °C, and subsequently washed and probed with species-specific secondary antibodies coupled to horseradish peroxidase  at room temperature for 2 h. For Methyl-PP2A-C protein detection, IP lysis buffer  was used instead of RIPA buffer, and the antibodies were probed in 0.5% nonfat milk in PBS-T. Immunoreactive proteins were detected by an enhanced chemiluminescence system as per the manufacturer’s protocol  or Kwik Quant Imager (Kindle Biosciences).2, 10% Glycerol) containing protease and phosphatase inhibitors and the lysates were incubated with anti-FLAG M2 beads for 2 h at 4 °C or used for V5 antibody pull-down assay. For B56a and B55a/d immunoprecipitations one to two 15 cm cell culture dishes with approximately 70% confluent cells or tumor tissues were lysed in lysis buffer (10% (v/v) glycerol; 135 mM NaCl; 20 mM Tris, pH 8.0; 1% Nonidet P-40; 1 mM PMSF; 0.03 U/ml aprotinin (Sigma), 1× Complete (Roche)) by 2″ sonication, centrifuged at 10956 × g at 4 °C for 10 min. Supernatant of lysates was adjusted to a protein concentration of 2 to 2.5 µg/µl. 1/10 of the lysate (input) was boiled in protein sample buffer and 1 ml of lysate was incubated either with B56α (1D3-E12) or B55α/δ (2G9) or B56α (ab89621) antibody crosslinked to protein G-Sepharose beads  to immunoprecipitate endogenous B55α/δ (clone 2G9) or B56α (clone 1D3-E12) for 1 h. The immune complexes were washed once with lysis buffer, 3× with Tris-buffered saline . For PP2A-A-V5 coimmunoprecipitation experiments, cells were harvested, and coimmunoprecipitation was performed per Dynabeads CoImmunoprecipitation protocol . V5 antibody (Bio-Rad) was coupled at a concentration of 7 μg/mg of Dynabeads. Cell lysate was incubated with the Dynabeads for 30 min, rotating, at 4 °C. Fresh conjugated beads were prepared for each biological replicates. The immunoprecipitate was boiled for 5 min at 95 °C in protein sample buffer. After that, SDS–PAGE, and immunoblotting was performed as described above. Densitometric quantification was performed using the Image J software and Image Lab Software.For immunoprecipitation (IP) experiments, nuclear or whole cell protein extracts were obtained from cells using NE-PER nuclear extraction kit  or RIPA buffer, respectively. The nuclear pellet was then lysed in an IP buffer by sonication. Nuclear lysates (0.5–2.0 mg) were pre-cleaned by incubation with protein G Dynabeads  for 1 h on a rotator at 4 °C. Next, an antibody (2–5 μg) was added to the pre-cleared lysates and incubated on a rotator at 4 °C overnight. The following day, Protein G Dynabeads were added to the nuclear lysate-antibody mix for 1 h. Beads were washed twice in IP or RIPA buffer containing 300 mM NaCl, resuspended in 40 μL of 2× loading buffer, boiled at 100 °C for 5 min, and subjected to SDS–PAGE and immunoblotting. For FLAG-tag immunoprecipitation, the cells were lysed in NP40 buffer  and a Tukey’s honestly significantly different (HSD) post hoc test for multiple comparisons, or unpaired Students The antibodies used in this study are given in Supplementary Data CDK7 inhibitor-THZ1 , R1881C-III , Trametinib , Enzalutamide , Apalutamide , Darolutamide , MLN4924 , Torin1 , LY294002 , MK-2206 , MG132 , DT-061 , Cycloheximide  and Rapamycin  were dissolved and aliquoted in DMSO . EGF  was dissolved and aliquoted in water.For Epidermal Growth Factor (EGF)  stimulation, the cells were plated in complete media at 60–70% confluency. After 24 h cells were washed with PBS, serum starved for 24 h and then treated with 100 nM EGF for the desired time points. Similarly, for androgen starvation, the cells were cultured in a medium without Phenol Red  and 10% charcoal-dextran stripped FBS  for the required time points post 24 h growth in a regular medium.For gene knockdown experiments, cells were seeded in six-well plates and transfected with 100–200 nM ON-TARGET plus SMART pool siRNA targeting S6K , β-TrCP  and non-targeting pool as a negative control  using lipofectamine RNAiMAX  according to the manufacturer’s instructions. The cells were then harvested 48/72 h post-transfection and used for the data presented.For the cell proliferation assay, the cells were seeded at a density of 50,000 per well in 12-well plates (n = 3). Twenty-four hours after seeding the cells were treated with ARD-69 or Enzalutamide or DT-061 or DMSO. Medium with the drug/compound was changed after every 48 h. The cells were harvested, and live cell numbers were counted in the conditions used by Countess II FL .n = 3) were seeded and treated with the required drugs/compounds or vehicle for 12-14 days. Media was replenished every 3–4 days. Colonies were fixed and stained using 0.5% (w/v) crystal violet  in 20% (v/v) methanol for 30 min, washed with distilled deionized water, and air-dried. After scanning the plate, the stained wells were destained with 500 μL 10% acetic acid and the absorbance was determined at 590 nm using a spectrophotometer . For VCaP or VCaP-Enzalutamide-resistant cells, viability was assessed by Cell-Titer GLO .For the colony formation assay, approximately 10,000 cells/well in six-well plates  supplemented with 0.1% Triton-X-100. After incubation on ice for 10 min, the nuclear pellet was collected by centrifugation at 1300 × g for 5 min at 4 °C, washed in Buffer A, and resuspended in Buffer B  with the same centrifugation settings, and incubated on ice for 30 min. The chromatin pellet was collected by centrifugation at 1700 × g for 5 min at 4 °C, washed and resuspended in Buffer B with 150 mM NaCl, and incubated on ice for 20 min. After centrifugation at 1700 × g for 5 min to remove proteins soluble in 150 mM salt concentrations, the pellet was then incubated in Buffer B with 300 mM NaCl on ice for 20 min and centrifuged again at 1700 × g to obtain the final chromatin pellet. The chromatin pellet was dissolved in a sample buffer, sonicated for 15 s, and boiled at 95 °C for 10 min. Immunoblot analysis was conducted on samples as described previously. All buffers were supplemented with Pierce protease inhibitor and Halt protease & phosphatase inhibitors.Chromatin-bound proteins were extracted following a protocol previously describedhttp://bioinfo.ut.ee/primer3-0.4.0/primer3) and synthesized by Integrated DNA Technologies. The primer sequences for the SYBR green assays qPCR can be found in the Supplementary Data The miRNeasy Mini Kit  was used to isolate total RNA from cells and the SuperScriptIV  kit was used to synthesize cDNA from 1 µg total RNA. qRT-PCR was performed using Fast SYBR Master Mix  or Taqman Fast Advance MMIX , and analyzed on QuantStudio3 . The ΔΔCt method was used to quantify target mRNA expression, as normalized to GAPDH transcript levels. Primers were designed using Primer3 Input (version 0.4.0) , pAR (1:100), and methyl C (1:50). Coverslips were washed three times with PBS-T and incubated with the respective Alexafluor secondary antibodies (1:200) for 1 h at room temperature followed by counterstaining with DAPI (Sigma). Coverslips were mounted on glass slides using VECTASHIELD PLUS Antifade Mounting Medium (Vector Laboratories) and cured overnight. Images were captured on a Zeiss LSM 880 confocal microscope. FIJI software was used to integrate the signal for quantitative image analysis. For specific quantification of nuclear staining, regions of interest were overlaid with the DAPI signal, and only co-localizing regions were included in the integration to exclude background cytoplasmic signals.2 according to the manufacturer’s instructions. The Phos-tag gels were washed with transfer buffer containing 4 mM EDTA for 20 min and then were washed with transfer buffer containing 0.1% SDS for 5 min before transfer onto PVDF membranes. The membranes were then immunoblotted with specific primary antibodies overnight followed by imaging and analysis.The protein samples were resolved on 6% PAGE gel containing 10 μM Phos-tag acrylamide AAL-107  and 50 μM MnCl2HPO4, and 2 mM KH2PO4 at 4 °C. Upon equilibration purified GST-tagged protein was loaded on the beads by incubating at room temperature for 45 min on a shaker. Following that 2–3 mg of nuclear protein, lysate was incubated with protein-bound glutathione beads at 4 °C for overnight on a shaker. The next day, the bead-bound complex was washed four times with PBS to remove unbound protein, and the bound proteins were eluted from beads by boiling the sample in SDS loading dye at 100 °C. Elute was resolved on SDS–PAGE and proteins of interest were detected using immunoblotting.Glutathione Sepharose beads  were equilibrated by incubating for 10 min (3×) in PBS -137 mM NaCl, 2.7 mM KCl, 8 mM NaFor in vivo ubiquitylation assay, HA- β-TrCP1, Flag-LCMT1, and His-ubiquitin expression plasmids were co-transfected into HEK293-T cells. After 36 h, 5 μM MG132  was added for 6 h before harvesting the cells. Transfected cells were lysed with 1% SDS and sonicated. The cell lysates were diluted with NP10 buffer and incubated with Ni-NTA beads at 4 °C for 2 h. Next, beads were washed three times with the NP40 buffer, boiled in 50 μl of 2× SDS loading buffer for 5 min, followed by western blot analysis with anti-V5-LCMT1 antibody to detect polyubiquitination.−1 Ubc3, 10 ng µl−1 Ubc5, 2.5 µg µl−1 ubiquitin (Boston Biochem), S6 kinase, and 1 µl of unlabeled in vitro transcribed/translated LCMT1 and/or βTrCP1. The reactions were incubated at 30 °C for 1 h, subjected to SDS–PAGE, then analyzed by immunoblotting with an anti-LCMT1 antibody.For in vitro ubiquitylation assay, the ubiquitylation of LCMT1 was performed in a volume of 10 µl, containing 50 mM Tris pH 7.6, 5 mM MgCl2, 2 mM ATP, 1.5 ng µl−1 E1, 10 ng µlhttps://www.agilent.com/store/primerDesignProgram.jsp), and Site-directed mutagenesis was performed using the QuikChange II XL site-directed Mutagenesis Kit  as per the manufacturer’s protocol. All the mutations were verified by Sanger sequencing.The pLX304-V5-LCMT1-WT plasmid was purchased from DNASU (HsCD00441432) and pLX304-V5-LCMT1-S8A, pLX304-V5-LCMT1-S9A, pLX304-V5-LCMT1-S12A, pLX304-V5-LCMT1-S8.9.12 A constructs were created from it through Site-directed mutagenesis. Similarly, pWZL hygro FLAG-HA TRAP220-WT  was used to create pWZL-HA-MED1-2A-a, pWZL-HA-MED1-2A-b, and pWZL-HA-MED1-2A-ab plasmids. Primers, shown in the Supplementary Data Other plasmids used in this study are V245 pCEP-4HA B56α , PPP2R1A plasmid in pLX304 , Flag-FBXW7, Flag- β-TrCP, pFN21K-AR-Halo, and MSCV-myrAkt (Addgene plasmid_ 49267-deposited by Lawrence Kane & Arthur Weiss), pLX304-LacZ , pLX304-EGFP , shNTC and shLCMT1 plasmids . The plasmids were transfected into HEK293-T or LNCaP cells using Lipofectamine 2000  as per the manufacturer’s protocol. The cells were then harvested 48/72 h post transfection and subjected to immunoprecipitation or immunoblotting or used for Lentivirus production. Lentiviral particles were generated in the lab at the University of Pennsylvania (UPenn) or in collaboration with the Vector Core at the University of Michigan. Following lentiviral production, viral particles were incubated on cells in penicillin/streptomycin-free media for 24 h, followed by media replacement with normal media. After 72 h, cells were selected in 16 µg/mL Blasticidin-Invivogen (pLX304 constructs) or sorted by FACS for GFP positivity (shLCMT constructs) to generate stable cell lines. Knockdown or overexpression of proteins was confirmed by immunoblotting.RNA-seq libraries were constructed using the TruSeq sample Prep Kit V2.5 (Illumina) according to the manufacturer’s instructions. In brief, 1 μg of purified RNA was poly-A selected and fragmented with a fragmentation enzyme. Poly-A selected/fragmented RNA templates were used to synthesize the first and second strands, and end-repair to PCR amplification was performed according to the manufacturer’s protocol. Libraries were purified and validated for appropriate size on a 2100 Bioanalyzer DNA 1000 chip . DNA library concentration was assessed using Qubit and normalized to 4 nM before pooling. Libraries were pooled in an equimolar fashion and diluted to a loading concentration of 1.8 pM. Library pools were clustered and run on the Nextseq500 platform (Illumina Inc.) with single-end reads of 75 bases.80 with default parameters to either the HG19 or MM10 reference genomes. To remove low-quality/duplicate reads and sort the aligned BAM files, Samtools (v1.13) was used. Filtered BAM files were converted to BigWig files to view on the UCSC browser using bamCoverage (v3.5.1) from the Deeptools suite. Cufflinks, Cuffquant, and Cuffnorm v2.2.181 were used to assemble, quantify, and normalize transcripts (FPKM) to assemble the count tables, respectively.Single-end reads were demultiplexed using Illumina’s bcl2fastq CLI tool. Fastqc was used to quality check the fastq files. Reads were then aligned using STAR v2.7.3.ahttps://bioconductor.org/packages/release/bioc/html/DESeq.html). DESeq282 performs internal normalization to account for library size and RNA composition bias provided the raw counts. Genes with a fold change of at least 1 and a q-value < 0.05 were considered to be differentially expressed.Differential expression analysis was done using the Bioconductor package, DESeq2 , LCMT1 overexpressing vs LAZ cells (LNCAP), and PTEN vs PTEN-Null (mouse organoids) using Hallmark signatures that are available in the Molecular Signatures Database (MSigDB) by BROAD institute.Gene enrichment analysis or GSEA is a tool that determines overrepresented genes in predefined gene sets that are biologically related using statistical methodsChromatin immunoprecipitation (ChIP) was performed using the iDeal ChIP-seq kit for Transcription Factors  according to the manufacturer’s instructions. In brief, cells were collected and subjected to DNA-protein cross-linking using formaldehyde. After cell lysis, the cross-linked chromatin was fragmented and immunoprecipitated using Protein A magnetic beads and antibodies targeting transcription factors of interest. IPure magnetic beads were used to purify eluted DNA, which then underwent library preparation and sequencing. Library preparation for Illumina NextSeq was performed using the NEBNext Ultra II DNA library prep kit (NEB) as per the manufacturer’s protocol. The quality of the library prepared was assayed using a BioAnalyzer-2100  and quantified using Qubit prior to loading of samples onto an Illumina NextSeq-500 to perform sequencing of 75 nucleotide paired-end reads at 40 million read depth.84. To overlap peaks and remove problematic regions known to produce enriched signals in several next-generation sequencing experiments, known as blacklists (https://github.com/Boyle-Lab/Blacklist), bedtools intersect (v2.30.0) was used.Paired-end reads were demultiplexed using Illumina’s bcl2fastq CLI tool. Fastqc was used to quality check the fastq files. Reads were then aligned using Bowtie2 (v2.2.5) with default parameters to the HG19 reference genome. MarkDuplicates from the genome analysis toolkit  was used to mark duplicates. To remove low-quality/duplicate reads and sort the aligned BAM files, Samtools (v1.13) was used. Filtered BAM files were converted to BigWig files to view on the UCSC browser using bamCoverage (v3.5.1) from the Deeptools suite. MACS2 (v2.2.6) was used to compute and find enriched peaks in BAM files83. Gene signature scores were computed using singScore85. Samples were split into those pre-/post-ARSI. The post-ARSI patients were further split based on high/low survival from the date of biopsy, and high/low time to progression on ARSI. Analysis of variance (Anova) and Wilcoxon signed-rank tests were used to determine differences between groups.From 4/2005-7/2021, 268 prostate cancer patients older than 18 years of age underwent tissue collection for tumoral RNA-sequencing at the University of Michigan . Clinical data including information on ARSI treatment regimens  and survival was collected from 05/2021-01/2022. RNA-sequencing data for 313 RNA libraries across 290 biopsies was processed using Turnkey Precision Oncology, a flexible in house computational pipeline. Gene signatures for androgen response and mTORC1 signaling were obtained from the Molecular Signatures DatabasePten knockout mice, we crossed Pbsn-Cre male mice Tg (Pbsn-cre) 4Prb/J  with Pten floxed mice . The genotype of the mice was confirmed using the primers provided in the Supplementary Data To generate the prostate-specific 86.Eight-week-old wild-type C5B6 and Pten floxed mice were euthanized and the prostate glands were dissected in DMEM 10% FBS. The prostate gland was transferred to a new petri dish with fresh dissecting media and minced with a razor blade. The minced tissue was digested with collagenase on a shaker at 37 °C for 2 h. Tissue chunks were spun down and digested with Trypsin/0.05% EDTA for 5 min at 37 °C. The cell/tissue suspension was passed through a 40 µm nylon mesh filter to get a single-cell suspension. 10,000 cells in 40 µL PrEGM media were mixed with 60 µL Matrigel and seeded to the rim of a 12-well plate, followed by adding 800 µL warm PrEGM in 30 min. In 10 days, both wild-type and Pten floxed P0 organoids were digested with Dispase and Trypsin into single-cell suspension and treated with Adeno-cre for 20 min to induce the knockout in the Pten floxed cells. Ten thousand treated cells in each group were seeded in 12-well plates, images were taken on day 10 and organoid sizes were measured using ImageJ. Genomic DNA and protein lysates were harvested on day 10 for the indicated molecular analysis2O2 for 5 min. After three times 5 min TBST washing, slides were blocked with blocking buffer (1.25% of goat serum) at room temperature for 1 h. Slides were applied with diluted primary antibody and incubated in a humidified chamber at 4 °C overnight. After three 10 min TBST washes, slides were incubated with secondary antibody  for 30 min at room temperature the color of the antibody staining was revealed by peroxidase-based detection  and the sections were counterstained with hematoxylin . Representative photographs were taken on a Keyence BZ-X Series All-in-one Fluorescence Microscope with 20x and 40x objectives. Images were assessed and quantified in ImageJ.Mouse prostate tissues were fixed in 4% of formaldehyde for 48 h, and paraffin-embedded through Molecular Pathology and Imaging Core at UPenn. Paraffin-embedded sections from the prostate of Pten floxed control and Pten knockout mice were deparaffinized with 3 changes of xylene for 5 min each. Slides were then rehydrated in 100% alcohol for 10 min, 95% alcohol twice for 10 min each, and 70% alcohol and distilled water for 10 min each. Slides were subjected to citrate-based (pH 6.0) antigen retrieval  at 95 °C for 30 min and followed by blocking endogenous peroxidase activity with 3% H6 VCaP cells stably overexpressing LCMT1 along with the control VCaP cells were injected into the dorsal flanks of the non-castrated NOD-SCID mice . The chi-square- or t-test was used to evaluate the association of individual tumor characteristics with engraftment.Eight million LNCaP expressing shNTC, shLCMT (knockdown study) were subcutaneously injected into the right flank or both the dorsal flanks of 5–8-week-old male non-castrated and castrated NCI/NOD SCID/NCr mice  in 50% Matrigel . Castration surgery was performed by Charles River before receipt of the mice. The tumor measurement study ended at 40 days, and mice with tumors were sacrificed due to body condition or used for subsequent analysis. Tumor tissue was harvested and was snap frozen in liquid nitrogen for immunoblotting analysis. Mice that did not form tumors at this time continued to be monitored for tumor growth and body weight for first detection tumor analysis, up to 80 days. Eight million LNCaP LacZ or LCMT1 (overexpression study) were subcutaneously injected into the right flank or both the dorsal flanks of 5–8-week-old male non-castrated NCI/NOD SCID/NCr mice . Similarly, 2 × 106 VCaP-EnzaR prostate cancer cells suspended in 80 μL of RPMI 1640 with 50% Matrigel were implanted subcutaneously into the dorsal flanks of the mice. When the tumor volumes reached approximately 80–90 mm3 mice were randomized into three treatment groups viz 15 mg/kg body weight DT-061, 50 mg/kg body weight DT-061 and vehicle-DMA (10%). The treatment was given orally b.i.d for 5 days a week for 3 weeks. In all studies tumors were monitored and recorded by digital caliper twice weekly, and animals were weighed twice weekly. The tumor volumes were estimated using the formula (π/6) (L × W2), where L = length and W = width of tumor. At the end of the treatment regimen the mice were sacrificed, and the tumors were extracted for further analysis.For in vivo efficacy studies of DT-061 2 × 10Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data