TP53. BRCA2 was nonsense-mutated (p.Q780*) and CREBBP was missense-mutated (p.P279R) in LuPanc-1. CDKN2A was missense-mutated (p.H83Y) in LuPanc-2. Notably, only LuPanc-2 harbored a partial or complete deletion of DPC4. LuPanc-1 cells exhibited high basal and transforming growth factor (TGF)-β1-induced migratory activity in real-time cell migration assays, while LuPanc-2 was refractory. Both LuPanc-1 and LuPanc-2 cells responded to treatment with TGF-β1 with the activation of SMAD2; however, only LuPanc-1 cells were able to induce TGF-β1 target genes, which is consistent with the absence of DPC4 in LuPanc-2 cells. Both cell lines were able to form spheres in a semi-solid medium and in cell viability assays, LuPanc-1 cells were more sensitive than LuPanc-2 cells to treatment with gemcitabine and FOLFIRINOX. In summary, both patient-derived cell lines show distinct molecular phenotypes reflecting their individual tumor biology, with a unique clinical annotation of the respective patients. These preclinical ex vivo models can be further explored for potential new treatment strategies and might help in developing personalized (targeted) therapy regimens.The prognosis of pancreatic ductal adenocarcinoma (PDAC) is exceedingly poor. Although surgical resection is the only curative treatment option, multimodal treatment is of the utmost importance, as only about 20% of tumors are primarily resectable at the time of diagnosis. The choice of chemotherapeutic treatment regimens involving gemcitabine and FOLFIRINOX is currently solely based on the patient’s performance status, but, ideally, it should be based on the tumors’ individual biology. We established two novel patient-derived primary cell lines from surgical PDAC specimens. LuPanc-1 and LuPanc-2 were derived from a pT3, pN1, G2 and a pT3, pN2, G3 tumor, respectively, and the clinical follow-up was fully annotated. STR-genotyping revealed a unique profile for both cell lines. The population doubling time of LuPanc-2 was substantially longer than that of LuPanc-1 (84 vs. 44 h). Both cell lines exhibited a typical epithelial morphology and expressed moderate levels of CK7 and E-cadherin. LuPanc-1, but not LuPanc-2, co-expressed E-cadherin and vimentin at the single-cell level, suggesting a mixed epithelial-mesenchymal differentiation. LuPanc-1 had a missense mutation (p.R282W) and LuPanc-2 had a frameshift deletion (p.P89X) in  Pancreatic cancer is currently the fourth most common cause of cancer-related deaths and is predicted to be the second leading cause of cancer-related mortality by the year 2030 . The majSurgical resection is the only curative treatment option for pancreatic cancer. However, only 10–20% of patients present with resectable tumors at the time of diagnosis, 30–40% present with a borderline resectable tumor, and 50–60% present with metastatic disease [The adjuvant treatment of patients with resectable PDAC by chemotherapy results in better overall survival. Modified combination therapy consisting of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (mFOLFIRINOX) resulted in a significantly better median overall survival of 54.4 months compared to that of gemcitabine alone (35.0 months) . HoweverCDKN2A (encoding the cyclin-dependent kinase inhibitor p16INK4) and DPC4, encoding SMAD4, a central mediator of transforming growth factor (TGF)-β signaling, has been implicated in modifying the response to gemcitabine or 5-FU [Overall, the 5-year survival rate of PDAC patients remains as low as 5–8% . The sel or 5-FU ,15,16. H or 5-FU . Taken tPreclinical models of PDAC are essential for drug development and the identification of predictive biomarkers. Primary, patient-derived cell lines are an indispensable tool among ex vivo models. Primary cell lines can provide valuable information on the phenotype and genotype of individual tumors, allow for functional assays of the individual tumor biology, and be used for the development of personalized treatment strategies by enabling individualized therapeutic drug testing ex vivo. However, the efficient ex vivo expansion of human carcinomas in tissue culture, especially that of PDAC, remains technically challenging. The Cancer Cell Line Encyclopedia (CCLE) from the Broad Institute comprises 60 pancreatic cell lines. Nevertheless, the full spectrum of the complex and diverse tumor biology of PDAC is not reflected sufficiently by these cell lines, and the clinical annotation and oncological outcome of the respective patient are mostly missing.Here, we describe two newly established patient-derived cell lines from surgical PDAC specimens named LuPanc-1 and LuPanc-2. Both cell lines were comprehensively characterized and tested with respect to the standard therapy regimens gemcitabine and FOLFIRINOX.TM with 4.5 g/L glucose (Gibco) supplemented with 20% (v/v) fetal calf serum (FCS) and 100 μg/mL streptomycin/penicillin. The cells were cultured at 37 °C in a humidified incubator with 5% CO2. The cells began to form colonies after about 7 d. Subsequently, the medium was replaced every 3–4 d. The number of cocultured fibroblasts was periodically reduced by differential trypsinization. After reaching stable growth of the cultured tumor cells, fibroblasts were depleted by an immunomagnetic assay based on fibroblast-specific antigens . Single-cell-derived clonal cultures were established from primary cultures by limited dilution, as previously described for the permanent PDAC cell line PANC-1 [® 21 System , according to the manufacturer’s instructions.The tissue was collected directly after the surgical resection of the tumor after approval by the University of Lübeck’s local ethics committee (# 16-281). The tissue was processed within 1 h after surgical resection and cut into 3–5 mm pieces using a scalpel. Pieces were dissociated using collagenase I  for 120 min at 37 °C. The cells were plated on tissue culture dishes in DMEM GlutaMAXe PANC-1 . The celGrowth curves for both cell lines were established by counting cells in three independent experiments every 24 h for 7 and 10 d for LuPanc-1 and LuPanc-2, respectively, after seeding 80,000 cells per well in a six-well plate. The final cell count was carried out after harvesting the cells using Accumax (PAN Biotech) in an improved Neubauer hemocytometer. The total cell number was determined by averaging the cell count in eight 1 mm squares. The cell numbers were normalized to the cell number initially counted 24 h after seeding.2. Extinction was quantified with a plate photometer, according to the manufacturer’s instructions. The excitation wavelength was set to 545 nm, and the emission wavelength was set to 600 nm. Each experiment was performed in triplicate and repeated three times.To measure the sensitivity to chemotherapy, 6000 cells of LuPanc-1 and 8000 cells of LuPanc-2 growing in a log phase were seeded per well in 96-well plates, and chemotherapeutics were added 24 h after cell seeding. For gemcitabine treatment, gemcitabine  was added to a final concentration in a range of 2.5–320 nmol/L for LuPanc-1 and 10–12,800 nmol/L for LuPanc-2. For FOLFIRINOX treatment, 5-FU, irinotecan, leucovorin, and oxaliplatin were combined, and the final concentration ranged as follows: 5-FU (Medac) 0.005–426 µmol/L, irinotecan (Medac) 0.07 nmol/L–5.78 µmol/L, oxaliplatin (GRY) 0.062 nmol/L–4.96 µmol/L, and leucovorin (Sigma Aldrich) 0.078 nmol/L–6.19 µmol/L. After 72 h, cell metabolism was measured by a Resazurin reduction assay. Resazurin (STEMCELL) was diluted in DPBS to a concentration of 10 mg/mL. This stock solution was further diluted in the medium, 20 µL was added per well to a final concentration of 40 µg/mL, and the cells were incubated for 2 h at 37 °C in a humidified incubator with 5% CO® Culture Slides; Corning, NY, USA and Sarstedt, Nümbrecht, Germany). The cells were permeabilized with 0.1% Triton-X and sequentially treated with goat serum, primary monoclonal mouse anti-human CK 7 antibody , primary monoclonal rabbit anti-human E-cadherin antibody , primary monoclonal mouse anti-human vimentin antibody , anti-Mouse Alexa Fluor® 594 secondary antibody , and anti-Rabbit Alexa Fluor® 488 secondary antibody . Appropriate negative controls were performed. Coverslips were mounted with Fluoromount-G medium including DAPI . The cells were visualized using a BZ-9000 (BIOREVO) fluorescent microscope  with a 20× PlanFluor EL NA 0.45 Ph1 objective lens under 0.285 s of exposure time for CK7 and 0.2 s of exposure time for E-cadherin and vimentin.The cells were cultivated for 72 h, with subsequent fixation with 4% paraformaldehyde in chamber slides -based assays were employed. For the Soft Agar-based assay, a total of 1000 viable single-cells were resuspended in 0.25% agar  and added on top of the precoated 48-well plate with 1.3% agar containing DMEM with 20% FBS. For the sphere formation assay in BME, 1000 cells were resuspended in 50 µL DMEM with 20% FBS, mixed with 150 µL Cultrex , and then plated on 48-well plates. During spheroid cultivation, the medium was replaced every 3 d. Sphere formation in both assays was monitored for 14 d. All experiments were performed in two independent biological replicates with three technical replicates each.® technology , as outlined in detail in previous publications [v/v) of collagen I and collagen IV (30 μL) to facilitate the adherence of the cells and thus enhance the duration of the signal recording. Each well was loaded with 60,000 (LuPanc-1) or 80,000 (LuPanc-2) cells in standard growth medium (see above). Cells were allowed to settle in the laminar flow hood for 30 min at RT, after which the assay was started and run for 24 or 48 h. Some assays were performed in the absence or presence of recombinant human (rh) TGF-β1  or the TGF-β type I receptor/ALK5 serine/threonine kinase inhibitor SB431542 . Data acquisition  and analysis were performed with the RTCA software .The migratory/invasive activities of LuPanc-1 and LuPanc-2 were determined with xCELLigenceications ,20. The DNA was extracted from both cell lines using the AllPrep DNA/RNA/Protein Mini Kit , according to the manufacturer’s instructions. Targets were enriched using the Agilent SureSelectXT kit, and libraries were prepared using the NEB Next Ultra DNA Library Prep Kit. Sequencing was performed on a NovaSeq 6000 using the v1.5 chemistry.fastp [Picard Tools (v2.18.4), and postprocessing was performed following GATK’s [Variant Effect Predictor , and annotations were converted into MAF format using vcf2maf (V1.6.21) (http://doi.org/10.5281/ZENODO.593251 (accessed on 24 April 2022); coverage for each variant was extracted directly from the vcf INFO field. The top 20 frequently mutated genes (FLAGS) [Sequencing data (paired-end fastq files) were trimmed  using fastp  (v0.23.0fastp  (v0.7.15g GATK’s . Single-mosdepth (v0.3.2) [Copy number aberrations were detected using Control-FREEC (v11.4) , and seq(v0.3.2) , resultiFor the Sanger sequencing of the KRAS locus, the following primers were used (5′–3′): forward: CCTTATGTGTGACATGTTCT; reverse: ATGGTCCTGCACCAGTAATA (amplicon size: 218 bp). Sanger sequencing was carried out as described previously .DPC4, SMAD2, SERPINE1, SNAI2, SOX2, TBP and GAPDH are given in The total RNA was isolated from LuPanc-1 and LuPanc-2 cells with an RNAeasy kit . A total of 2.5 μg of RNA of each sample was reverse transcribed  with 200 U of M-MLV Reverse Transcriptase and 2.5 μM random hexamers. Target gene expression was quantified by qPCR on an I-Cycler  with Maxima SYBR Green Mastermix  and normalized to the expression of either TATA box-binding protein (TBP) or GAPDH. The PCR primer sequences for We established two patient-derived primary pancreatic cancer cell lines from PDACs (LuPanc-1 and LuPanc-2). LuPanc-1 cells were derived from a PDAC of a 64-year-old male patient who presented to our surgical department with a locally advanced tumor of the pancreatic tail with infiltration of the stomach. The patient underwent distal pancreatectomy, splenectomy, and simultaneous partial resection of the stomach. The histopathological assessment of the resected tumor revealed a moderately differentiated ductal adenocarcinoma of the pancreas . The patient received adjuvant chemotherapy with a reduced dose of FOLFIRINOX due to intolerance. The patient developed a local recurrence and peritoneal carcinomatosis 9 months after the surgical resection of the primary tumor and subsequently received palliative chemotherapy with gemcitabine/nab-paclitaxel.LuPanc-2 cells were derived from a locally advanced PDAC of a 63-year-old female patient. The patient presented with a tumor located in the pancreatic corpus with local infiltration of the portal vein and celiac trunk. The patient underwent an extended multiorgan pancreatic corpus and tail resection (Appleby procedure) at our department. The histopathological assessment of the resected tumor revealed a poorly differentiated ductal adenocarcinoma of the pancreas . The patient received adjuvant chemotherapy with FOLFIRINOX but developed a retrogastric local recurrence 8 months after the resection of the primary tumor. The patient subsequently received palliative radiotherapy.For both cell lines, cells were isolated immediately after the surgical resection of the tumor by mechanical and enzymatic dissociation of a representative specimen. After 7 days of cultivation, first, adherent colonies of epithelial tumor cells surrounded by fibroblasts appeared, as shown exemplarily for LuPanc-1 in Both established cell lines were authenticated by STR genotyping for 21 loci . No idenFirst, we determined the general growth behavior of both cell lines. The population doubling times of LuPanc-1 and LuPanc-2 cells were estimated to be 44 h and 84 h, respectively, as measured by manual cell counting, indicating that LuPanc-2 cells grew substantially slower than LuPanc-1 cells A. To furThe aggressiveness of pancreatic cancer cells is associated with the acquisition of mesenchymal characteristics in comparison to those cells that are well-differentiated and epithelial-like . Thus, wTP53 had a missense mutation in LuPanc-1 (p.R282W) and a frameshift deletion (p.P89X) in LuPanc-2 (BRCA2 was nonsense-mutated (p.Q780*) in LuPanc-1. CDKN2A (encoding p16INK4) had a missense mutation (p.H83Y) in LuPanc-2 and CREBBP had a missense mutation (p.P279R) in LuPanc-1. We further specifically analyzed KRAS, DPC4, RNF43, ARID1A, TGFBR2, GNAS, RREB1, and PBRM1, which have been described to be recurrently mutated in PDAC by the Cancer Genome Atlas Research Network [KRAS, BRCA2, CDKN2A, and DPC4 as well as gains of RNF43, GNAS, and PBRM1 in both cell lines. For ARID1A, we detected a loss in LuPanc-1 but a gain in LuPanc-2. Notably, a complete loss of DPC4  and a loss of RREB1 were found in LuPanc-2 but not LuPanc-1 cells, while, conversely, LuPanc-1 but not LuPanc-2 cells showed a loss of TP53 and a gain of CREBBP. KRAS codons 12 and 13 in the WES data, we specifically analyzed these loci by subsequent Sanger sequencing and detected a codon 12 mutation  in both cell lines.We profiled the genomic landscape of both cell lines by whole-exome sequencing (WES). After the removal of commonly mutated genes without any effect, 135 and 76 mutations were detected in LuPanc-1 and LuPanc-2, respectively. Most of the mutations were missense mutations in both cell lines, followed by nonsense mutations . MutatioLuPanc-2 A. BRCA2  Network , but nonThe mutational signatures and, in particular, the presence of mutant KRAS suggested that both cell lines possess a transformed phenotype. To scrutinize this functionally, we performed sphere formation assays, exploiting the ability of cancer cells to grow and generate visible colonies (spheres) from single-cells when cultured in a semi-solid medium. As expected, both cell lines generated large numbers of spheres in soft agar and Matrigel, with LuPanc-2 cells forming substantially more spheres than LuPanc-1 cells .DPC4 (encoding SMAD4) and TGFBR2 (encoding the type II receptor), have been described to be recurrently mutated in PDAC and to be associated with increased or decreased resistance to gemcitabine [SERPINE1 , SNAI2 (encoding SNAIL2/SLUG), or SOX2, respectively , which represents a non-invasive and label-free approach for the continuous  monitoring of cell migration and invasion on a cell culture level . For LuPHaving shown above that LuPanc-1 cells have retained sensitivity to rhTGF-β1 with respect to the activation of Smad proteins and invasion-associated target genes, we sought to know whether rhTGF-β1 would also impact the cells’ migratory behavior in vitro. To this end, this growth factor increased the migratory activity over that of control cells . The IC50 for gemcitabine was 13.6 nM (IC90 of 50.5 nM) for LuPanc-1 and 357.1 nM for LuPanc-2 (IC90 of 2812 nM)  was 573.8 nM (IC90 of 5659 nM) for LuPanc-1 (50 (or IC90) for FOLFIRINOX since we were unable to obtain a normal dose–response curve.As described above, gemcitabine and FOLFIRINOX are essential chemotherapeutics used in clinical routine practice for PDAC treatment. Accordingly, the sensitivity of both cell lines was tested in Resazurin-based viability assays for gemcitabine monotherapy and FOLFIRINOX by determining the respective half-maximal inhibition concentration (IC2812 nM) . The IC5PDAC is a highly malignant carcinoma with an exceedingly poor prognosis. The overall 5-year survival rate of PDAC patients is as low as 5–8% . SurgicaPreclinical studies using patient-derived cell lines may be useful in predicting the response of a particular patient to gemcitabine/FOLFIRINOX or for the development of alternative and novel treatment strategies. Several studies have described specific molecular subtypes of PDAC based on transcriptional profiles ,11,12. TIn this study, we established two novel PDAC-derived cell lines named LuPanc-1 and LuPanc-2 from the surgical specimens of PDAC patients. Both cell lines presented with a transformed phenotype, as evidenced by their mutational spectra and their ability to form spheres in semi-solid media, also suggesting that the cells have stem cell properties. Clinical follow-up was annotated for both patients, and both corresponding cell lines were characterized comprehensively at the molecular level and functionally with respect to invasive behavior and the response to chemotherapeutic drugs.Primary, patient-derived cell cultures are a highly relevant tool because they reflect the in vivo state and physiology better compared to immortalized secondary cell lines. Both cell lines established in this study were taken into the primary culture directly from surgical specimens. They can be maintained in the culture without changes in cell dynamics and a loss of viability, obviating the need for cell immortalization ,44. MostFinally, one of the main drawbacks in the design of personalized treatment strategies is the marked inter-tumor heterogeneity in PDAC. As mentioned above, although the CCLE database already contains 60 pancreatic cancer cell lines, these include histological entities other than PDAC, and those within the PDAC fraction are likely to not represent the full spectrum of PDAC histomorphological subtypes. Moreover, a substantial number of those cell lines have been derived from metastases rather than the primary tumor, which means that these do not necessarily respond in the same way to chemotherapeutic drugs as the cancer cells from the patient’s primary tumor. With respect to histomorphological differentiation, LuPanc-1 cells present with a mixed phenotype co-expressing both epithelial and mesenchymal markers at a single-cell level, as demonstrated for E-cadherin and vimentin by the immunofluoresecence and immunoblot analysis of single-cell-derived clonal cultures. This phenotype has so far only been described for the PANC-1 cell line and has been shown to reflect the presence of subpopulations of cells with different EMT phenotypes . This mi50 357.1 nM) compared to LuPanc-1 (IC50 13.6 nM). LuPanc-1 showed a similar response to gemcitabine compared to other commonly used cell lines such as CAPAN-1 (11.5 nM), BxPC-3 (18 nM), MIA PaCa-2 (36–40 nM), and PANC-1 (50 nM) [We also determined the response of both cell lines to gemcitabine and FOLFIRINOX, which are the relevant treatment regimens in current clinical care. LuPanc-2 was substantially more resistant to gemcitabine (IC 50 nM) ,50,51,52 nM ,50,5 (50 nM) . SurprisBRCA2 nonsense mutation might offer a rationale for an alternative treatment strategy based on PARP inhibitors such as olaparib.Both patients who gave rise to our cell lines received additive therapy with FOLFIRINOX after surgery but developed a local recurrence in a short postoperative interval of 9 and 8 months, respectively. The patient from whom LuPanc-1 was derived received gemcitabine treatment for the local recurrence, which is in line with a good response of the derived cell line in vitro. However, the detected The epithelial-mesenchymal phenotyping of both cell lines showed that while the phenotype of LuPanc-2 cells was tentatively epithelial based on the presence of CK7 and E-cadherin, along with the absence of vimentin protein expression, LuPanc-1 cells combined both epithelial and mesenchymal traits, revealing a mixed E/M phenotype. Cells that co-express markers of both subtypes have been postulated to possess a higher stem cell potential and cellular plasticity. Tumor cells with this mixed phenotype, which is also referred to as partial or hybrid EMT, have been shown to be more metastatic , which iDPC4 in LuPanc-2 cells by CNV analysis, which was subsequently confirmed by SMAD4-specific qPCR. DPC4 has been described to be recurrently mutated in PDAC, and its genomic deletion was significantly correlated positively with disease progression [DPC4 was associated with an altered sensitivity to treatment with gemcitabine [DPC4 status and the gemcitabine response in this panel of PDAC cell lines, among DPC4 isogenic cell lines, DPC4-defective cells were about twofold less sensitive to gemcitabine than wildtype cells. A correlation analysis for IC50 values and TGF-β pathway activities in these isogenic cells confirmed a negative correlation with TGF-β pathway activities [50 in LuPanc-2 cells. However, it should be mentioned that, in another study, both SMAD4-deleted PDAC cell lines and patient-derived organoids exhibited a higher sensitivity toward gemcitabine (and other cell cycle-targeting agents) due to the upregulation of cell cycle-related genes [INK4 inactivation were three- to fourfold less sensitive to gemcitabine [CDKN2A, we speculate that the combined alterations in CDKN2A and DPC4 are causal for the high gemcitabine IC50 of LuPanc-2 cells. Another interesting issue is whether the inability of LuPanc-2 cells to exhibit basal migratory activity in vitro is a direct consequence of their unresponsiveness to TGF-β1 or the SMAD4 loss, i.e., due to the failure of autocrine TGF-β to stimulate cell motility.Of particular interest was the finding of the deletion of gression  and negagression . In addicitabine ,15,57. Acitabine . Despitetivities , matchined genes , suggestcitabine . Since LCell lines and, in particular, primary patient-derived cell lines are an essential tool for preclinical drug stratification and can help in developing personalized treatment regimens ,60. Howe


The inhibition of poly(ADP-ribose) polymerases (PARPs) and ataxia telangiectasia and Rad3-related (ATR) would be an alternative approach for cancer treatments. The aim of this study is to investigate the synergy of the different combinations of PARP inhibitors  and ATR inhibitor AZD6738. A drug combinational synergy screen that combines olaparib, talazoparib, or veliparib with AZD6738 was performed to identify the synergistic interaction, and the combination index was calculated to verify synergy. TK6 isogenic cell lines with defects in different DNA repair genes were used as a model. Cell cycle analysis, micronucleus induction, and focus formation assays of serine-139 phosphorylation of the histone variant H2AX demonstrated that AZD6738 diminished G2/M checkpoint activation induced by PARP inhibitors and allowed DNA damage-containing cells to continue dividing, leading to greater increases in micronuclei as well as double-strand DNA breaks in mitotic cells. We also found that AZD6738 was likely to potentiate cytotoxicity of PARP inhibitors in homologous recombination repair deficiency cell lines. AZD6738 sensitized more genotypes of DNA repair-deficient cell lines to talazoparib than to olaparib and veliparib, respectively. The combinational approach of PARP and ATR inhibition to enhance response to PARP inhibitors could expand the utility of PARP inhibitors to cancer patients without BRCA1/2 mutations. Genomic instability is one of cancer hallmarks . Cancer It has been hypothesized that DNA damage response (DDR) pathways mediated by ataxia-telangiectasia mutated (ATM) and ATR are responsible for cell survival in response to PARP inhibitors . There iIn this study, we investigated effects of ATR inhibitor AZD6738 in potentiating the cytotoxicity of three PARP inhibitors using different DNA repair-deficient TK6 isogenic cell lines as a model. We show that combining PARP inhibitors with AZD6738 contributes to an increase in replication-associated DNA damage and micronucleus induction. We also reveal that a synergistic effect is likely to be observed in homologous recombination repair deficiency cell lines. AZD6738 specifically synergizes some PARP inhibitors in particular DNA repair-deficient cell lines, providing rationale for scrutinizing synergistic interactions between PARP and ATR inhibitors in immortalized or cancer cell lines that have defects in another HRR-associated gene.in vitro experiments required mock treatment at specific concentrations of DMSO if the final concentration of DMSO was higher than 0.1%.Three PARP inhibitors and one ATR inhibitor were purchased from the MedChemExpress, LLC: olaparib (cat. no. 763113-22-0), talazoparib (cat. no. 1207456-00-5), veliparib (cat. no. 912444-00-9), and AZD6738 (cat. no. 1352226-88-0). All drugs were reconstituted and stored in dimethyl sulfoxide . Some WT), tumor suppressor p53-binding protein 1 knockout (−/−53BP1), RAD54 knockout (−/−RAD54), ATM knockout (−/−ATM), and breast cancer type 1 susceptibility protein conditional knockout (AID/AIDBRCA1) human cell lines [2. The AID/AIDBRCA1 cell line was engineered by tagging the miniauxin-induced degron (mAID) into the C-terminal end of the BRCA1 gene [BRCA1 gene expression for in vitro experiments, AID/AIDBRCA1 cells were cultured in the medium containing 250 μM 3-indoleacetic acid  for 2 h before exposing to drugs and for entire experiments.Human lymphoblastoid TK6-derived wild-type  alone, 0.45 μM AZD6738 alone, or combinations of each PARP inhibitor and AZD6738 for 20 h. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.3% Triton-X  for 10 min at room temperature. DNA content was stained by propidium iodide  and assessed by flow cytometry on a Navios flow cytometer and Kaluza analysis 2.1 software . At least 10,000 events were analyzed per sample.Asynchronized cells were treated with indicated PARP inhibitor  alone, 0.45 μM AZD6738 alone, or combinations of each PARP inhibitor and AZD6738 were used to treat asynchronized cells for 20 h before harvesting. Cells were harvested onto glass slides using a Shandon Cytospin® 4 cytocentrifuge . A cell staining protocol for micronuclei and serine-139 phosphorylation of the histone variant H2AX (gH2AX) focus formation assays has been described previously [PARP inhibitor  alone, AZD6738 alone, or combinations of each PARP inhibitor and AZD6738 for 72 h. For the combination of PARP inhibitor and AZD6738, the drug combinations were serially plated with constant ratio concentration in 96-well plates. Cell viability was determined by CellTiter-Glo® Luminescent Cell Viability Assay . The combination index (CI) values at IC50 were calculated using CompuSyn version 1.0 .Asynchronized cells were seeded into 96-well plates at a final concentration of 30,000 cell/ml in 200 p values were calculated using Student's t-test. For gH2AX focus formation assay, medians were shown, and p values were calculated using the Mann-Whitney test. For CI calculation, averages and standard errors were displayed.Statistical analysis was performed using GraphPad Prism software version 8 . All results except gH2AX focus formation assay and CI calculation were expressed as averages and standard deviations, and WT and the other DNA repair-deficient cell lines. The increased G2/M population was obviously observed in −/−ATM and AID/AIDBRCA1 cell lines treated with PARP inhibitor alone. These data suggest that olaparib, talazoparib, and veliparib induce G2/M arrest. In contrast to PARP inhibition, the treatment of AZD6738 alone decreased the G2/M population in every indicated genotype compared to control treatments. Moreover, combining olaparib, talazoparib, or veliparib with AZD6738 significantly decreased the G2/M population in every indicated genotype compared to PARP inhibitor treatment alone , −/−ATM (CI = 0.605 ± 0.05), and AID/AIDBRCA1 cell lines (CI = 0.63 ± 0.06). For the combination of olaparib and AZD6738, the synergistic interaction was observed in both −/−ATM (CI = 0.59 ± 0.01), and AID/AIDBRCA1 cell lines (CI = 0.57 ± 0.12), while combining veliparib with AZD6738 showed the synergy in only the AID/AIDBRCA1 cell line (CI = 0.55 ± 0.10)  . These rBRCA1/2 genes. In addition to BRCA1/2, several somatic mutations in genes associated with the HRR pathway, such as ATM, BAP1, and PALB2 genes, have been recognized [BRCA1/2. However, the impacts of DNA repair dysfunction on response to the combination of PARP and ATR inhibitors are not fully understood.Nowadays, several PARP inhibitors have been approved for cancer patients. However, most cancer patients who are prescribed the PARP inhibitor have to have germline mutations in cognized . Both recognized . In addicognized . Previoucognized , 21. TheWT cell lines when combining each individual PARP inhibitor with AZD6738. It indicates that AZD6738 tends to potentiate PARP inhibitors in HRR-deficient cells. A significantly increased number of micronuclei at basal levels in RAD54-, ATM-, and BRCA1-deficient cell lines, which show the synergistic interaction between some types of PARP inhibitors and AZD6738, are also observed. However, in a 53BP1-deficient cell line, a number of micronuclei at basal levels are significantly elevated (Supplementary Table This study showed that AZD6738 abolishes the G2/M checkpoint activated by PARP inhibitors, leading to the increases in both micronuclei and DNA damage that is consistent with a previous study . Though WT, 53BP1-, and RAD54-deficient cell lines. Moreover, the combination of talazoparib and AZD6738 shows the synergistic interaction in RAD54-, ATM-, and BRCA1-deficient cell lines except WT and 53BP1-deficient cell lines. However, veliparib in combination with AZD6738 is exclusively synergistic in a BRCA1-deficient cell line. This finding suggests that cancers with DNA repair deficiency due to the mutations in HRR pathway-associated genes apart from BRCA1/2 could synergistically respond to the specific combinations of PARP and ATR inhibitors, and the combination strategy of PARP and DDR inhibitors could probably be expanded to treat other DNA repair-deficient cancers besides BRCA1/2-deficient cancers. Nevertheless, the reasons why some combinations of PARP inhibitors and AZD6738 show the synergism in specific HRR-deficient cell lines are unclear. For example, talazoparib, which is 100-fold more potent than olaparib, while veliparib is the weakest one [This study showed novel opportunities for PARP inhibitors by combining with DDR inhibitors in DNA repair-deficient cell lines. Mechanistically, the synergistic interaction of AZD6738 combined with PARP inhibitors is similar to a previous study . Althougkest one , shows t50 values of AZD6738 among different genotypes, even though an increased number of micronuclei were noticed in DNA repair-deficient cell lines, especially in a BRCA1-deficient cell line. According to the reported IC50 values of AZD6738 from this study (Supplementary Table This study also showed that there is no considerable difference in ICBRCA1/2. Moreover, PARP inhibitor dosages, especially talazoparib whose side effects are nearly similar to conventional chemotherapeutic drugs [For a clinical implication, the synergism demonstrated in this study supports the rationale to further investigate the combination of PARP and ATR inhibitors in HRR-deficient cells beyond BRCA1/2 that may give advantages for cancer patients without germline mutations in ic drugs , could bIn conclusion, this work emphasizes the value of investigating the possible indications of the combination of PARP and ATR inhibitors for cancer treatments to enhance the efficacy of PARP inhibitors and expand their use to other groups of cancer patients. This work demonstrates the evidence that supports the development of cancer therapy through the combination strategy of PARP and ATR inhibitors, and assesses HRR deficiency as predictive biomarkers to identify individuals tending to respond to PARP and ATR inhibitor combination treatments. The use of DDR inhibitors to synergistically potentiate PARP inhibitors could be an alternative option for cancer patients in the future.


O-mannosyl glycosylated, and β-DG, an integral transmembrane polypeptide. α-DG is involved in the maintenance of tissue integrity and function in the adult, providing an O-glycosylation-dependent link for cells to their extracellular matrix. β-DG in turn contacts the cytoskeleton via dystrophin and participates in a variety of pathways transmitting extracellular signals to the nucleus. Increasing evidence exists of a pivotal role of DG in the modulation of normal cellular proliferation. In this context, deficiencies in DG glycosylation levels, in particular those affecting the so-called matriglycan structure, have been found in an ample variety of human tumors and cancer-derived cell lines. This occurs together with an underexpression of the DAG1 mRNA and/or its α-DG (core) polypeptide product or, more frequently, with a downregulation of β-DG protein levels. These changes are in general accompanied in tumor cells by a low expression of genes involved in the last steps of the α-DG O-mannosyl glycosylation pathway, namely POMT1/2, POMGNT2, CRPPA, B4GAT1 and LARGE1/2. On the other hand, a series of other genes acting earlier in this pathway are overexpressed in tumor cells, namely DOLK, DPM1/2/3, POMGNT1, B3GALNT2, POMK and FKTN, hence exerting instead a pro-oncogenic role. Finally, downregulation of β-DG, altered β-DG processing and/or impaired β-DG nuclear levels are increasingly found in human tumors and cell lines. It follows that DG itself, particular genes/proteins involved in its glycosylation and/or their interactors in the cell could be useful as biomarkers of certain types of human cancer, and/or as molecular targets of new therapies addressing these neoplasms.Dystroglycan (DG) is a glycoprotein composed of two subunits that remain non-covalently bound at the plasma membrane: α-DG, which is extracellular and heavily  DAG1 gene, which is transcribed into a single 5.7 kb mRNA whose protein product exhibits 895 amino acids (aas) and a molecular mass of 97.5 kDa in our species. This polypeptide undergoes post-translational cleavage in an autocatalytic fashion at residue Ser-654 to yield two subunits, α-DG and β-DG, which remain non-covalently associated [O-linked sugar chains, among them O-mannosyl glycans, and also containing three N-linked glycosylation sites [O-glycosylation of its core polypeptide, α-DG is detected in western blots as a broad band ranging in size between 100 and 190 kDa, depending on the tissue under study [N-glycosylated residue and lacking O-glycosylation sites [Dystroglycan (DG) was discovered in the embryonic chicken brain as the predominant plasma-membrane glycoprotein able to bind laminin , the majsociated –6 , which links ECM proteins such as laminin and proteoglycans to the actin cytoskeleton via dystrophin in skeletal muscle cells and non-muscular tissues –19. Thise domain , 22–25. e domain , 27. Thiskeleton .DAG1 gene), a set of known or putative glycosyltransferases and other enzymes, mostly residing in the endoplasmic reticulum or the Golgi complex , FKRP, LARGE1 and many others [SLC35A1 [INPP5K [TRAPPC1 and GOSR2 [O-mannosyl glycan structure harbored by α-DG is composed of three core glycans, named M1, M2 and M3, of which the ECM-binding motif, designated as matriglycan, is added to the phosphorylated core M3 glycan [n that is synthesized by the enzyme products of genes B4GAT1 and LARGE1 [To date, 22 genes have been identified which encode, in addition to DG itself DAG gene, a  pathway , 29–32. y others , of whic[SLC35A1 , INPP5K  [INPP5K , TRAPPC1nd GOSR2  have bee3 glycan . Such mod LARGE1 . In humad LARGE1 –44. Fromd LARGE1 , 41–43.Mycobacterium leprae indirectly through its ability to bind laminin, allowing the entrance of these pathogens into human cells [In addition to muscle and neural cells, DG is expressed in many other cell types, all of which share the property of being in direct contact with basement membranes in their residing tissue , 45. Manan cells , 49, 50.an cells , 51–53, an cells , 55, as http://gco.iarc.fr). The most frequently diagnosed tumor types from higher to lower incidence are breast, lung, colorectum, prostate, stomach, liver, esophagus and cervix. Altogether, they represented ca. 57% of the global incidence cancer cases in 2020 and together with pancreas cancer they constitute the top nine most deadly cancers in the world today, being lung cancer the most frequent cause of cancer death in males and breast cancer in females [It is estimated that over 19 million new cancer cases and nearly 10 million cancer deaths occurred in 2020 worldwide according to the Global Cancer Observatory  and inflammatory state (driven by cells of the immune system), which in turn promote tumor progression by diverse mechanisms . The mos2 for their sustenance, together with an ability to clear metabolic wastes and CO2. Such needs are fulfilled by the tumor-associated neovasculature, which is generated by a process of angiogenesis [Another hallmark capability of cancer cells is their ability to evade the actions of tumor-suppressor genes. Most prominent among the regulators disrupted in cancer cells are the retinoblastoma/RB protein and p53, two prototypical transcription factors playing a pivotal role in regulatory circuits determining the cells’ decisions to proliferate or, alternatively, undergo senescence and apoptosis , 60. Sinogenesis .Regarding mechanisms underlying invasiveness, carcinomas arising from epithelial tissues can progress to achieve higher pathological grades of malignancy, reflected in local invasion and distant metastasis. Cancer cells may alongside develop alterations in their shape and in their ability to attach to other cells and the ECM . The expO-mannosylated carbohydrate structures on α-DG forming part of matriglycan (such as IIH6 and VIA4-1) for both western blotting and immunohisto/cytochemistry analyses. These alterations have been reported in a wide variety of tumor types and cell lines, and have been associated with tumorigenesis contributed by loss of the cells’ link to the ECM [During cancer progression, primary tumor cells escape from their normal growth control by modifying the binding affinities of their cell membrane receptors. In a wide sense, unregulated proliferation and invasion are often accompanied by dramatic alterations in the expression pattern of molecules active at the interface between the ECM basement membrane and the cell’s plasma membrane , 66. In  the ECM , 70, 71. the ECM , 55. In  the ECM , 73, thi the ECM , 52. Add the ECM , 74. AltIn this work we review current literature on the relationship between DG and cancer, which has noticeably declined over the last decade. This review intends to compile all the existing information on alterations detected in the expression and function of DG and the genes/proteins involved in its glycosylation in human tumors and tumor-derived established cancer cell lines. In addition to the involvement of deficiencies in α- and/or β-DG expression, and α-DG glycosylation , in the pathogenesis of cancer, alterations in the expression levels of particular DGP-associated genes and proteins are discussed in the light of the specific tumor and cell-line types in which they have arisen. Finally, possible implications of these findings in the unraveling of new molecular biomarkers and potential therapeutical targets of cancer are emphasized in a biomedical context.DAG1 mRNA, α-DG and/or βDG protein levels, and/or low or undetectable glycosylated α-DG levels, which are compiled in Table A significant number of human primary tumors of many different types have been found to exhibit low or undetectable DG expression, in terms of DAG1 gene has been analyzed at the mRNA level, and has been found undetectable compared to its levels in normal oral squamous epithelium [In squamous cell carcinomas of the oral mucosa, the expression of the ithelium . Also, uithelium , 79–81. DAG1 mRNA levels between normal and tumor tissue. However, a complete loss of α-DG glycosylation  has been found in nearly all samples studied, with a large reduction in glycosylated α-DG levels in the rest of them [In addition to oral carcinomas, alterations in DG have been found in other tumors of the digestive system. In esophageal cancer, no significant differences were observed in  of them . Also, a of them , 97.Undetectable α-DG matriglycan levels have been reported in primary gastric cancer, together with reduced expression of the β-DG subunit . FurtherDAG1 mRNA, that was concluded not to be statistically significant, which suggested that altered post-transcriptional modifications, such as glycosylation, might result in defective protein processing or sorting [As in other tumors of the digestive system, almost undetectable glycosylated α-DG and β-DG protein have been found in pancreatic ductal carcinoma, together with a replacement of β-DG membrane localization by cytoplasmic staining . It must sorting . It was  sorting , 84.Other studies have unveiled that in colon cancer α-DG glycosylation is nearly absent and β-DG is underexpressed compared to healthy tissue , 86, 97.Among tumors of the reproductive system, prostate cancer constitutes one of the most studied regarding DG. By using an antibody to the core protein of α-DG, it was concluded that this polypeptide was underexpressed (by −5.26-fold) in samples from elderly prostate cancer patients, and the same accounted for β-DG . In otheIn other types of tumors of the reproductive system, such as cervical and vulvar cancers, hypoglycosylation of α-DG has also been detected . A progressive, significant decrease of the percentage of cells exhibiting glycosylated α-DG has been observed in correlation with the degree of tumor progression in squamous intraepithelial lesions and invasive cervical carcinomas, as compared to the fraction of cells positive for glycosylated α-DG quantitated in normal cervical epithelium . The samDAG1 mRNA was found to be downregulated (by −2.17-fold) in clear cell renal cell carcinoma [In studies of tumors of the urinary tract, the arcinoma . Furthemarcinoma , 91, 92,arcinoma . Conversarcinoma .In breast cancer, a progressive reduction of α-DG matriglycan and β-DG expression has been reported in correlation with increasing tumor grade and stage . Also, iRegarding tumors of the nervous system, diminished immunoreactivity from glycosylated α-DG (by ‒2.62-fold) and from β-DG protein (by ‒0.60-fold) has been reported in malignant, grade III and IV gliomas, the latter also designated as glioblastomas , 94, 95.DAG1 mRNA was found to be downregulated (by −2.17-fold) in leukemia primary blasts with different degrees of maturation , as compared to control (CD34+) stem-progenitor cells from healthy individuals [In tumors of the hematopoietic system, DG alterations have been studied in patients with acute myeloid leukemia (AML). Here, the ividuals . An undeividuals .In tumors of the musculoskeletal system, a reduction in glycosylated α-DG levels has only been found in Ewing’s sarcoma (a rare cancer developing in bones or surrounding soft tissue) and rhabdomyosarcoma , although in the latter α-DG (core) protein levels were not found altered, and β-DG was found expressed at different sizes, of 43 kDa and higher , 96. By Given the wide variety of tumors in which the levels of expression and/or glycosylation of DG have been found altered, investigations have been carried out in a large number of model cell lines representative of such variety of tumors, in order to better understand the role of DG in cancer progression. Table Regarding tumors of the digestive system, several oral squamous cell carcinoma (SCC)-derived cell lines have been analyzed, with the result that glycosylated α-DG was undetectable , 81. At DAG1 mRNA was found to be normally expressed [DAG1 mRNA expression [In other studies, it was determined that the esophageal adenocarcinoma cell line FLO-1 showed a reduction in the levels of α-DG matriglycan and β-DG expression, although the xpressed . Also, αxpressed , 103. Fixpressed , 104. Thpression , 100.DAG1 mRNA, which led to the proposal of a post-transcriptional regulatory mechanism being operative in these cell lines [Among tumors of the reproductive system, there exists a wide variety of studies on prostate cancer cell lines. In the highly-metastatic prostate cell line PC3, as well as in the less-metastatic cell lines LNCaP and DU145, α-DG matriglycan levels were found to be undetectable , 105, dell lines . Noticeall lines , 105. Inll lines , 109, alll lines , 109.Regarding the cervical cancer HeLa cell line, although the α-DG core protein was detected, its glycosylation levels were found reduced . Yet, β-DAG1 mRNA expression, with normal levels of the latter being detected, again suggesting regulation at the post-transcriptional level [To date, breast cancer is the tumor type most studied regarding DG alterations in cell lines. Glycosylated α-DG was found to be undetectable or nearly absent together with virtually undetected β-DG levels in MCF7 , 106, T4al level . Regardial level , 85, 100al level .Undetectable expression levels of α-DG core protein in parallel to α-DG hypoglycosylation have been found in cell lines derived from human glioblastomas, namely U87MG and A172MG , 106. AlDAG1 gene has been found to be downregulated at the mRNA level in cell lines derived from tumors of the hematopoietic system. Also, decreased expression levels were observed in the HL-60 and Kasumi-1 leukemia-derived cell lines of α-DG core protein , and especially of β-DG (by ‒ 6.25- and ‒ 2.17-fold), correlating with an underexpression of DAG1 mRNA (by ‒ 2.50- and ‒ 1.49-fold) [The 49-fold) , 85. In 49-fold) .In the melanoma cell lines BLM, M14 and IF6, glycosylation of α-DG was reported to be nearly absent . Higher DAG1 mRNA and/or α-DG (core) protein in a limited number of reports, and in conjunction with underexpression of β-DG protein in most cases. The implications of these findings in the light of both α-DG and β-DG involvement in tumorigenesis are discussed below.Summarizing results above, α-DG has been found deficiently glycosylated in a wide variety of human tumor samples Table  and cancO-mannosyl glycosylation pathway can be seen in Fig. An altered expression at the mRNA and/or protein level(s) of genes associated with dystroglycanopathies, responsible for α-DG glycosylation, has also been evidenced in a variety of human primary tumors. The steps at which these genes and their protein products act in the α-DG POMT1 and POMT2 genes, which encode protein O-mannosyltransferases 1 and 2, two enzymes that act by forming a heterocomplex to carry out the first step in the O-mannosyl glycosylation of α-DG using dolichol-phosphate-mannose as a donor [POMT1 and POMT2 mRNA levels were determined in primary blasts derived from patients with AML. The results obtained revealed that in AML samples these genes were overexpressed  in comparison to healthy individuals [POMT2 mRNA was underexpressed in breast tumor samples, which correlated with a reduced relapse-free survival and increased tumor aggressiveness [The expression of  a donor , 130, ha a donor . Also, Pividuals . Additiosiveness .DOLK gene, encoding the enzyme dolichol kinase, has been found overexpressed (by 5.42-fold) at the mRNA level in breast cancer samples, especially from patients with triple-negative breast cancer (TNBC) [https://www.ncbi.nlm.nih.gov/geo), accession no. GSE38959. In addition, the expression of genes DPM1, 2 and 3, encoding the three subunits of dolichol-phosphate mannosyltransferase complex, has been found altered in several tumor types. From several databases it has been determined that genes DPM1 and 2 are overexpressed, at the mRNA level, in head and neck squamous cell carcinoma, and esophagus, colon, rectum and hepatic cancers , while DPM3 is observed overexpressed in esophagus, liver (by 1.67-fold) and breast cancers, as well as in glioblastoma [The r (TNBC) , as seenblastoma . These tblastoma .POMGNT1 gene, encoding protein O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, which is the next enzyme in acting after POMT1 and 2 by branching the α-DG O-glycosylation pathway in competence with the POMGNT2 gene product enzyme [POMT1/2, the POMGNT1 gene was found upregulated at the mRNA level in renal cell carcinoma (by 1.20-fold) [The t enzyme  Fig. 2)POMGNT1 gt enzyme . Overexpt enzyme , 115. Fu20-fold)  and prim20-fold) .POMGNT2 gene, encoding protein O-mannosyl β-1,4-N-acetylglucosaminyltransferase 2, has been found reduced (by ‒ 2.22-fold) in kidney cancer [POMGNT2 gene was found underexpressed at the mRNA level, alike six of the other genes, whereas the remaining three genes were found overexpressed. This expression pattern has been found in patients with high-risk uveal melanoma and has been correlated with a low survival and a higher probability of developing metastasis [B3GALNT2 gene, encoding β-1,3-N-acetylgalactosaminyltransferase 2, have been reported in cancer, being upregulated both its mRNA and protein product (the latter by 1.31-fold) in samples of hepatocellular carcinoma, and its levels directly correlating with tumor size and with a lower survival of patients with this tumor type [B3GALNT2 mRNA has been found overexpressed (by 2.56-fold) in breast cancer samples, especially from patients with TNBC [B3GALNT2 was detectably downregulated in rhabdomyosarcoma [The expression of y cancer . Also, ty cancer . The POMtastasis . Alteratmor type . Furtherith TNBC , althougosarcoma  and kidnosarcoma  biopsy sPOMK gene (formerly SGK196), encoding protein O-mannose kinase, have also been found in different types of breast cancer, compared to healthy breast tissue, by analyzing the data compiled in the Gene Expression Profiling Interactive Analysis  and Oncomine (https://www.oncomine.org/resource/login.html) microarray databases [N-glycosylated  and lacking N-glycosylation in both tumor and healthy tissues. It was determined, however, that in basal-type breast cancer (BLBC) POMK is only present in its N-glycosylated form, its overexpression being related with a better relapse-free survival from this subtype of tumor [N-glycosylated POMK promoted an increased phosphorylation of the AKT serine/threonine kinase, with ensuing activation of the PI3K/AKT/glycogen synthase kinase-3β (GSK-3β)/Snail signaling pathway, involved in metastasis, while the expression of α-DG core protein remained essentially unaltered [POMK has been found slightly downregulated (by ‒ 1.23-fold) in human biopsy samples [Increased levels (by 1.56-fold) of the mRNA of the atabases . Overexpof tumor . Also, inaltered . Regardi samples .FKTN) has been found to be related to the tissue of origin or the progression of tumors in patients with stomach cancer [B4GAT1 gene (formerly B3GNT1) has been detected underexpressed at the mRNA level in pancreatic ductal adenocarcinoma, this being associated with a poor patient prognosis [FKRP and LARGE1, encoding fukutin-related protein and LARGE xylosyl- and glucuronyltransferase 1, respectively, have been reported in AML primary blasts , at variance with genes POMT1, POMT2 and POMGNT1, which were found overexpressed in cells of this tumor type (see above) [LARGE1 mRNA expression data, retrieved from the Oncomine database, have been compared between cancerous and healthy tissues of different types. A deficient expression of LARGE1 was detected in colon adenocarcinoma (by ‒ 1.30-fold), prostate cancer (by ‒ 1.61-fold), infiltrating urinary-bladder cancer (by ‒ 1.85-fold), breast carcinoma (by ‒ 1.20-fold), glioma (by ‒ 2.63-fold) and melanoma (by ‒ 1.45-fold) [LARGE1 gene was originally identified at a chromosomal region frequently deleted in human meningiomas [LARGE1 gene has been reported in patients with primary glioblastoma, this being related to an increase of Ki-67+ cells and a shortened survival [LARGE1 mRNA was observed in squamous cell carcinoma of head and neck [https://portal.gdc.cancer.gov) was used to identify possible changes in the mRNA expression of genes involved in α-DG glycosylation in colon and kidney carcinomas from different patients [LARGE1, data collected in this database on advanced colorectal cancer (CRC) samples reflected elevated levels of LARGE2 mRNA. This is a LARGE1 paralog so far not found mutated in any DGP cases, but whose encoded enzyme catalyzes the same reaction , was evidenced in patients with renal cell carcinoma, which was associated with an increased mortality [LARGE2 (by ‒ 3.15-fold) has also been reported in prostate cancer [An upregulation at the mRNA or protein level of the gene encoding fukutin  . Separat45-fold) , as well45-fold) , 106, rh45-fold)  and non-45-fold) . Interesingiomas  that, siingiomas . Consequsurvival . On the and neck  and hepaand neck , being aand neck . Finallypatients . At varie latter . On the rly ISPD ), encodinsferase , 120, anortality . The reae cancer .The expression of genes and proteins associated with DGPs has also been analyzed in a wide variety of human tumor-derived cell lines. Table POMT1 mRNA has been reported to be downregulated as a consequence of promoter hypermethylation in the HCT116 cell line of colon cancer, whereas normal colon mucosa cells lacked POMT1 hypermethylation [POMT2 gene (but not POMT1) has been found underexpressed in the breast cancer cell lines MCF7, MDA-MB-231, MDA-MB-253 and T47D, in keeping with observations in samples from patients with breast cancer [POMT2 has been found downregulated at the mRNA and protein levels in the undifferentiated gastric carcinoma cell line Kato III, in a coherent fashion with reduced global protein O-mannosyl glycosylation in this cell line and in human gastric cancers in general [POMT1 and POMT2 genes have also been analyzed, together with POMGNT1, in the HL-60 and Kasumi-1 leukemia cell lines. These three genes were concluded to exhibit a reduced expression in both cell lines , although at a difference with samples from patients with AML, where the three were detected to be overexpressed [The hylation . The POMt cancer . In addi general . The mRNxpressed .DPM1, 2 and 3 genes are overexpressed , at the mRNA level in the SMMC-7721 cell line. Also, and at variance with DPM2 and 3, the DPM1 mRNA was upregulated (by 1.50-fold) in the Hep G2 hepatic cancer cell line, which suggests that DPM1 could be a potential biomarker with survival prognostic value in patients with hepatocellular carcinoma [Coherently with results reported in patients with liver cancer, arcinoma .B3GALNT2, it has been found upregulated at both mRNA (from 1.08- to 2.85-fold) and protein (from 1.51- till 2.97-fold) levels in all hepatocellular carcinoma cell lines analyzed, with the sole exception of BEL-7405, where a reduced expression (by ‒2.56-fold) of B3GALNT2 was observed at the mRNA, but not at the protein level [Regarding in level . Additioin level .FKTN gene has been observed at the mRNA level in the HL-60 and Kasumi-1 leukemia cell lines , again in contrast to the overexpression exhibited by patients with AML [FKTN mRNA was found upregulated in the stomach cancer cell lines MKN-1 and MKN-45, and at the protein level additionally in the HSC-57 cell line, which was consistent with the overexpression also reported in samples from patients with stomach cancer [FKTN, the FKRP mRNA was found underexpressed in the HL-60 and Kasumi-1 cell lines , which was in keeping with the results from patients with AML [A reduced expression of the with AML . Undetecwith AML . On the h cancer . Alike Fwith AML .B4GAT1 gene have been found to be decreased or undetectable in cell lines derived from prostate cancer, including PC3, PC3-L and LNCaP, and from breast cancer, such as MDA-MB-231 and MDA-MB-435 [O-mannosyl glycosylation pathway  [LARGE1 mRNA has been detected in the hepatic cancer cell lines Huh7 (by 6.20-fold), Hep G2 (by 4.40-fold) and SMMC-7721 (by 7.50-fold), in correlation with cell growth, proliferation, migration, invasiveness and cell cycle [LARGE2 gene have been detected in the PANC-1 and BxPC-3 cell lines of pancreatic cancer, TEM4-18 of prostate cancer, A498 of renal cell carcinoma, and MDA-MB-231 and ZR-75-1 of breast cancer [The mRNA levels of the A-MB-435 . Its enccer line  and in acer line , 87, 106arcinoma , in the arcinoma  (Table 467-fold) , in the 67-fold) , and in 67-fold) . Also co67-fold) . Howeverll cycle . Finallyt cancer , whereast cancer .DOLK, DPM1/2/3, POMGNT1, B3GALNT2, POMK and FKTN and/or their encoded proteins, all of which (except fukutin) act at early steps of the α-DG O-mannosyl glycosylation pathway rendering core M1-M3 glycans , acting at earlier steps of the α-DG glycosylation process, is apparent instead, exerted (among other mechanisms) through the modulation of intracellular signaling pathways, as also explained in the next sections. Regarding leukemia, controversial data have been provided between AML tumors and cell lines for mRNA levels of most of DGP-associated genes, whose expression additionally has not been addressed at the protein level, which precludes to draw consistent conclusions and calls for further investigations.Trying to summarize results above, if only human solid tumors and their derived cell lines are taken into account, it can be concluded that genes see Fig. . HoweverConsidering data obtained so far on alterations in the glycosylation and/or expression of DG and DGP-associated genes, responsible for α-DG glycosylation, a correlation has been underlined between such alterations, especially hypoglycosylation of α-DG, and the progression (tumor grade and/or stage), severity and poor prognosis of cancer. Such relationship has been experimentally documented, as mentioned above, in gastric , pancreaIn the light of the effect brought about by α-DG hypoglycosylation on tumorigenesis, analyses have been made on laminin binding in a number of human established cancer cell lines, mainly derived from prostate carcinomas. All the prostate cancer cell lines studied so far that exhibited hypoglycosylated α-DG in terms of matriglycan levels Table  had concLARGE2 gene [LARGE2 expression inversely correlated with tumor progression, from a localized to an invasive stage, in a tissue cDNA microarray organized according to prostate cancer stage. It followed that LARGE2 functionally glycosylated α-DG, thereby reducing the cells’ invasive and proliferative potential. At variance, in the PC3-L cell line it was documented that its virtual lack of laminin-binding glycans and enhanced cell migration was associated with a reduced expression of the B4GAT1 gene  are directly correlated with the mRNA levels of the GE2 gene . FurtherC3 cells . This inC3 cells , 139. AlC3 cells , 141, asLARGE1 gene has been determined to be a primary cause of loss of laminin binding by DG. This defect was attributed to an epigenetic phenomenon in the HeLa (cervix), MDA-MB-231 (breast) and H1299 and H2030 (lung) cell lines, where in all cases the LARGE1 promoter was found hypermethylated [LARGE1 expression and  α-DG matriglycan levels were recovered, this rescuing their ability to bind laminin and decreasing their migratory ability [LARGE1 expression, since an underexpression of other genes involved in α-DG glycosylation has been documented in some of these cell lines, namely the mRNAs of genes POMT2, this likely due to epigenetic regulation exerted by the histone methyltransferase EZH2 [B4GAT1 [LARGE2 in MDA-MB-231 cells [+ cells), likely by inhibiting activation of the mitogenic transcription factor, c-Jun [In human rhabdomyosarcoma biopsies  and a nuthylated , as it athylated . In this ability , 81. Howase EZH2 , B4GAT1  [B4GAT1  and LARG31 cells , 103 and31 cells   gene, ess role , and it ess role , 63, 145ess role , 103. Thess role . In thisO-mannosyl glycosylation are found overall, and especially of E-cadherin, which is both detectably underexpressed and poorly O-glycosylated, this leading to a partial cytoplasmic mislocalization of the latter with loss of cell-cell adhesion capability [MGAT5 mRNA, encoding the N-acetylglucosaminyltransferase named GnT-Va, and reduced levels of POMT2 mRNA and protein. These and other findings support the notion that a coordinated interplay exists between O-mannosyl glycosylation and N-glycosylation positively and negatively modulating, respectively, E-cadherin function, and that loss of the first in favor of the second could lead to loss of E-cadherin suppressive functions in cancer, thus contributing to tumor progression and metastasis [In samples of patients with esophageal cancer a loss of α-DG matriglycan has been detected together with a downregulation of β-DG Table  and E-capability . This octastasis .DAG1 (cDNA) was overexpressed in T4-2 cells, cell rounding  was elicited by laminin, cell polarity was restored and their tumorigenic potential reduced by increasing the levels of PTEN [Separately, studies of the expression of E-cadherin and α-DG glycosylation (matriglycan levels) have been conducted on breast cancer cell lines, where it was found that lack of polarized cell growth only took place in those cell lines exhibiting negative α-DG glycosylation and/or expression together with negative E-cadherin levels . This wa of PTEN , a well  of PTEN .α-DG expression and its functional glycosylation have also been detected on tumorigenic glioma stem cells (GSCs) residing in the perivascular bed of the tumor and in the most aggressive mesenchymal-like (MES-like) GBM tumor compartment . Within Finally, in breast cancer, although a correlation between α-DG hypoglycosylation and loss of their ability to laminin anchoring has been demonstrated in many cell lines , 106, a POMT1/2, POMGNT2, CRPPA, B4GAT1, LARGE1/2 and/or their encoded proteins are consistently downregulated, with very few exceptions , and their tumorigenic action must thus be exerted instead through modulation of signal transduction or gene expression.In human solid tumors and derived cell lines, genes s Tables ,  4. Sined genes , 141. ThCDH1 and LARGE2 genes, as compared to the less-aggressive PC3-E+ (prostate) cell line, which exhibited both molecular phenotypes [ZEB1 and LARGE2 genes through examination of clinical samples deposited in the TCGA database. It was found that mRNA levels of both genes inversely correlated between them in four types of cancer: prostate adenocarcinoma, clear cell renal cell carcinoma, invasive breast carcinoma and lung adenocarcinoma, in which DG expression and/or functional glycosylation were downregulated [LARGE2 expression by directly binding to its promoter [B4GAT1 and LARGE1 genes existing in prostate cancer cell lines, as it was also the case for breast cancer cell lines  is frequently inactivated by mutation, which allows Wnt activation and the concomitant nuclear translocation of β-catenin [LARGE2 intron 1 and activates its mRNA expression, with ensuing enhancement of LARGE2-dependent matriglycan synthesis on α-DG. This results in an increase of α-DG laminin-binding capacity and, associatedly, in a reduction of CRC cells migration and invasiveness [It has also been determined that cell lines lacking both α-DG matriglycan and E-cadherin expression, such as TEM4-18 (prostate), A498 (kidney) and MDA-MB-231 (breast), displayed a high mRNA expression of the gene encoding the EMT-promoting, zinc-finger transcription factor ZEB1 together with a downregulation of enotypes . Human tegulated . Experimpromoter . Howeverpromoter . This co-catenin , 154. Thsiveness . α-DG glsiveness , 105, asO-mannosyl glycosylation possibly mediated by both POMGNT1 and GnT-Vb, the latter being highly expressed in the brain [POMGNT1 silencing decreases cell growth and invasion in human glioblastoma cell lines [POMGNT1 knockout in the human HEK293T cell line also leads to a lower proliferation and migration rates, in a fashion associated with loss of cell-laminin interaction and increase of cell-cell adhesion mediated by N-cadherin [CDH2 gene) found together with a quantitatively and qualitatively altered N-cadherin N-glycosylation, possibly related to a mildly-decreased MGAT5 expression, encoding the GnT-Va enzyme. Also, a sustained activation of the ERK/MAPK pathway was demonstrated, reflected by increased levels of phosphorylated ERK1/2, p38 MAPK and MEK, together with overexpression of a set of EMT-promoting transcription factors, including Snail [POMGNT1 D179Rfs*11 mutation, this contributing to explain some of his/her clinical DGP symptoms. These results are in keeping with the link  between classic O-mannosylation and N-glycosylation of E-cadherin pointed out above, with changes in the first impacting on the second, unveiled in human gastric carcinomas [On the other hand, little is known on the mechanisms underlying the pro-oncogenic action of several DGP-associated proteins, since they have been documented experimentally so far in a limited number of instances of human tumor types and cancer-derived cell lines. In this light, POMGNT1 clearly exerts a pro-oncogenic role in glioblastoma, likely by inhibiting the intrinsic phosphatase activity of receptor-type protein tyrosine phosphatase β/ζ (RPTPβ/ζ) through its he brain , 155–157he brain . This nehe brain , 159. Coll lines . In thiscadherin . This wang Snail . These arcinomas  and durircinomas .N-glycosylation status appears to be more relevant than its expression levels for its tumor-suppressing (anti-invasion and anti-metastasis) capacity and hence for a better patient prognosis [N-glycosylated POMK has been evidenced to interact with signaling proteins (other than α-DG) to inhibit the AKT/GSK-3β/Snail metastatic/EMT pathway in breast cancer cells [N-glycosylation in breast tumor tissue remains to be evidenced. In an opposite fashion, B3GALNT2 appears to act in breast cancer in a secreted, N-glycosylated form by indirectly promoting FGF2-elicited angiogenesis mediated by GnT-Va [N-glycosylated) by indirectly increasing the activity of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine [FKTN/fukutin mRNA and protein in gastric cancer [FKTN mRNA in hepatic cancer [Regarding POMK, although found overexpressed in breast cancer, a mechanism thought to play a protective role especially in BLBC, its rognosis . In thiser cells  preventier cells , and  and matriglycan synthesis on α-DG are frequently reduced in many different types of primary tumors Table , these cAccumulating evidence indicates that β-DG is a functionally-versatile protein normally exerting a number of functions from its location at the plasma membrane as part of the DGC, and also inside the nucleus. At the plasma membrane β-DG serves as a multifunctional scaffold able to bind, by virtue of its cytoplasmic domain, to various proteins involved in different signal transduction pathways , 53. TheDAG1 cDNA a decrease of tumorigenicity was achieved in the prostate cancer LNCaP cell line without a recovery of α-DG matriglycan levels [Given that β-DG is often found fragmented, and its expression reduced or undetected in many human carcinomas, these alterations can have different effects on the role exerted by β-DG on different signal transduction pathways, resulting in the growth-rate and metabolic changes observed in tumorigenic cells , 97, 100n levels . This stIn addition to its critical roles in cell adhesion, signaling and cytoskeletal remodeling played at the plasma membrane, β-DG is often found in the nucleus. To reach this compartment, β-DG undergoes endocytosis from the plasma membrane, in a process independent of α-DG ligand binding and that is positively modulated by its phosphorylation on Tyr-892, which disrupts its interaction with dystrophin , being tDAG1 transcript, the immunolocalization pattern of α-DG and β-DG in the plasma membrane was very similar when comparing control (CD34+) cells from healthy individuals with primary blasts from AML patients or with the HL-60 and Kasumi-1 cell lines [POMT1, POMT2, POMGNT1, FKTN, FKRP and LARGE1 genes in the two cell lines  and ETV1 (ETS translocation variant 1) [ETV1 expression (by siRNA transfection) strongly compromises the invasive capacity of both androgen-dependent (LNCaP) and -independent (C81) cells, suggesting that ETV1 plays an important role in prostate cancer metastasis [ETV1, which originates by chromosomal rearrangement, is capable of activating the Ras/Raf/MAPK pathway in the absence of activated, phosphorylated ERK [ETV1 transcription and, in turn, of MAPK-dependent signaling leading to an increase in cell migration, could represent a positive feed-back mechanism driving prostate cancer progression [β-DG proteoforms of 43 kDa and smaller in size have been detected in both the cytoplasmic and nuclear fractions of healthy and prostate tumor tissue . Howeverg motifs , and heng motifs , 99 or ag motifs . In thistastasis . Furtherated ERK . Therefogression , this meFinally, the proposal has been raised by our group , 175 andA deficient or undetectable glycosylation of α-DG is consistently found in a wide variety of human tumor types and cancer cell lines. Also, a correlation exists with very few exceptions between hypoglycosylation of α-DG and loss of laminin binding, and thereby of cell anchoring to the ECM of the tissue in which the tumor originates. Although this is not clearly a causative or initial event during the origin of the tumor, it is a significant factor contributing to tumorigenesis and the malignant phenotype. Whether cause or consequence, it leads to an increase in cell growth and proliferation, together with the acquisition of migration and invasion properties, eventually resulting in metastasis and malignancy. Additionally, β-DG anomalous downregulation, fragmentation and/or translocation to the nucleus is increasingly clear to contribute to the tumorigenic process.DAG1 mRNA and/or α-DG (core) protein. However, in an ample set of human tumors and cell lines α-DG hypoglycosylation has been linked to downregulation of genes associated with DGPs or their encoded proteins, which are enzymes participating in the complex process of α-DG O-mannosyl glycosylation. Indeed, knockdown of some of these genes, including POMT1, B4GAT1 and LARGE1, or of the DG-encoding gene itself, DAG1, results in a loss of laminin-binding glycans with a concomitant increase in cell migration involving the activation of AKT and ERK. Conversely, forced overexpression of DAG1 rescues laminin-binding ability in a number of cell lines, with inhibition of cell-cycle progression, loss of anchorage-independent growth and decrease of tumorigenicity. Downregulation of DGP-associated genes in tumors and cell lines has been attributed to their silencing through promoter methylation or to transcriptional repression, as it has been demonstrated for LARGE1 and LARGE2 genes, respectively. It is thus concluded that hypoglycosylation of α-DG is an event associated with tumor origin and/or progression, and likely necessary for the detachment of cancer cells from their ECM.In a limited number of studies low levels of glycosylated α-DG  have been demonstrated to parallel an underexpression of O-mannosyl glycosylation, in favor of aberrant N-glycosylation mediated by GnT-Va. The silencing of the E-cadherin encoding gene, CDH1, is mediated by the EMT-promoting transcription factors ZEB1 and Snail, two regulators that are overexpressed in many cancers, and which act jointly to repress both CDH1 and LARGE2 mRNAs. This translates into reduced glycosylated α-DG and E-cadherin levels, with consequent loss of cell-cell contacts, a feature of abnormal, adult EMT that has been associated with invasiveness and tumor metastasis. In this context, it is known that DG is lost together with E-cadherin during metastatic spread, and that both molecules are reexpressed during a process resembling MET when colonizing a secondary target organ.A relationship has also been found between α-DG hypoglycosylation and loss of both E-cadherin expression and POMGNT2, CRPPA, B4GAT1, LARGE1 and LARGE2 and/or their encoded proteins, are found downregulated , by molecular mechanisms that are beginning to emerge, as discussed above, and that are in need of further research.There are some types of tumors such as kidney, liver, breast, and AML in which several DGP-associated genes are usually found to be altered at the same time Tables  and 4. WWhile α-DG interaction with laminin is important for cellular polarization and maturation, β-DG itself plays a function as a signaling molecule and indirect modulator of gene expression independently of α-DG glycosylation, aimed at regulating cell proliferation, which must be taken into account in the field of cancer pathogenesis. As part of the DGC, β-DG acts as a scaffold or multifunctional adaptor in the cytoplasm, binding to and sequestering Grb2 and other signaling proteins of the Ras/Raf/MAPK pathway, and eventually bringing about inhibition of cell division-stimulatory signals and/or enhancement of growth-inhibitory signals, thereby playing a role itself as a tumor suppressor. However, the β-DG tumorigenic potential becomes evident when it is found underexpressed or fragmented, by MMPs and other unidentified proteases, into lower-molecular-weight proteoforms, of which the ∼31 and (the full-length) 43 kDa species are found phosphorylated in the nucleus. Interference of normal cytoplasmic signal transduction by these β-DG species results in dysregulation of cell proliferation and differentiation by not well known mechanisms. Also, β-DG forms assemblies at the nuclear envelope and matrix, acting as a scaffolding protein involved in the organization of nuclear architecture and the modulation of transcriptionally-active regions as a resident component of different intranuclear compartments. It is thus tempting to suggest that, when dysfunctional, β-DG could act to downregulate DGP-associated genes in some human cancers, this resulting in decreased α-DG matriglycan levels and ensuing enhanced cell proliferation. The transcription factors with whom β-DG interacts physically or functionally to modulate the cell division cycle or cell differentiation remain essentially unknown, with the exception of ETV1, a transcriptional regulator with an involvement in prostate cancer metastasis.LARGE1 and LARGE2 could be potential targets for therapies enhancing their levels. Also, strategies should be investigated aimed at increasing DG expression, and hence α-DG glycosylation function in cell-cell and cell-ECM adhesion and β-DG tumor-suppressor function in a joint fashion. The unraveling of all interacting partners of α-DG and β-DG at their different locations in and outside the cell should eventually constitute a source of new targets of therapies against human cancers. Finally, virtually all the genes and proteins dealt with in this review should be included in NGS- or microarray-based transcriptomic panels or protein microarrays aimed at detecting alterations in their expression in human tumor samples with diagnostic and/or prognostic value.To conclude, the panorama of α-DG glycosylation and β-DG role as preventers of cell detachment from the ECM, uncontrolled cell division and invasiveness, as shown in Fig.


Prostate cancer is a common cancer in men that annually results in more than 33 000 US deaths. Mortality from prostate cancer is largely from metastatic disease, reflecting on the great strides in the last century of treatments in care for the localized disease. Metastatic castrate resistant prostate cancer (mCRPC) will commonly travel to the bone, creating unique bone pathology that requires nuanced treatments in those sites with surgical, radio and chemotherapeutic interventions. The bone morphogenetic protein (BMP) pathway has been historically studied in the capacity to regulate the osteogenic nature of new bone. New mineralized bone generation is a frequent and common observation in mCRPC and referred to as blastic bone lesions. Less common are bone destructive lesions that are termed lytic.We queried the cancer genome atlas (TCGA) prostate cancer databases for the expression of the BMP pathway and found that distinct gene expression of the ligands, soluble antagonists, receptors, and intracellular mediators were altered in localized versus metastatic disease. Human prostate cancer cell lines have an innate ability to promote blastic‐ or lytic‐like bone lesions and we hypothesized that inhibiting BMP signaling in these cell lines would result in a distinct change in osteogenesis gene expression with BMP inhibition.We found unique and common changes by comparing these cell lines response and unique BMP pathway alterations. We treated human PCa cell lines with distinct bone pathologic phenotypes with the BMP inhibitor DMH1 and found distinct osteogenesis responses. We analyzed distinct sites of metastatic PCa in the TCGA and found that BMP signaling was selectively altered in commons sites such as lymph node, bone and liver compared to primary tumors.Overall we conclude that BMPs in metastatic prostate cancer are important signals and functional mediators of diverse processes that have potential for individualized precision oncology in mCRPC. During signal transduction, BMP ligand dimers bind to type I and type II serine/threonine receptor monomers, resulting in the formation of a hetero‐tetrameric kinase complex. Following ligand‐receptor formation, the constitutively active type II receptors phosphorylate the type I receptors to activate the latter's kinase domains.BMP signaling is associated with a wide variety of human cancers, including prostate, breast, colorectal, lung, ovarian, and others.The BMP pathway has long been studied in prostate cancer due to the role of BMPs in the unique prostate cancer tropism to metastasize in bone. In prostate cancer, BMP signaling has been directly correlated with tumor development and metastasis. For example, BMP6 was detected in over 50% of prostate samples from metastatic adenocarcinoma patients, while expression was not observed in non‐metastatic tumors or benign prostate tissue.Metastatic prostate cancers have a unique propensity to colonize bone, where they most often exhibit an osteoblastic phenotype characterized by increased osteoblast activity and bone lesions. The high affinity of prostate cancer for bone is the result of multiple interactions between the tumor and pre‐metastatic site in a non‐cell autonomous manner.In our study, we demonstrate the nuanced expression of BMP signaling using recent online data from cBioPortal and the Cancer Cell Line Encyclopedia (CCLE).22.15 in each well of a six well tissue culture plate. Once cells reached a confluence of ~90% they were treated with 10 μM DMH1 in DMSO (Selleckchem), for 24 h and RNA was collected.The PC‐3 , 22Rv1 , DU145 , VCaP , and C4‐2B  cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM)  high glucose with sodium pyruvate (Corning), 10% fetal bovine serum (FBS)  (Corning) and Antibiotic  and Antimycotic Solution  (Sigma‐Aldrich). The MDA PCa 2b  was obtained from the ATCC and cultured in F‐12K Medium  with 20% FBS (Corning), Cholera Toxin , mouse Epidermal Growth Factor (EGF) , O‐Phosphoethanolamine , Hydrocortisone , Sodium Selenite , human recombinant insulin  and Antibiotic and Antimycotic Solution (Sigma‐Aldrich). All cell lines were routinely tested for mycoplasma infection by PCR and authenticated with the short tandem repeat analysis done by the University of Colorado Cancer Center Cell Technologies Shared Resource. Cells were plated at 5.0 × 102.22 Profiler™ PCR Array Human Osteogenesis  was used to assess the expression of 84 genes associated with osteogenesis. The cDNA was synthesized using the RT2 Profiler™ PCR Array protocol. Genes that were ±2‐fold change were validated with multiple samples. The iScript cDNA Synthesis Kit and protocol  was used to generate cDNA from 500 ng of total RNA. Real‐time PCR reactions were performed using the SsoAdvanced Universal SYBR Green Supermix  on a CFX QPCR instrument (Bio‐Rad). The targets and primers used are listed in Figure 2 Profiler™ blastic‐like cell line results was purchased from Qiagen (GeneGlobe). All genes were run in technical and biological quadruplicate, with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as the housekeeping gene to normalize gene expression.The RT2.3p value of .05 for significance and were one‐tailed Mann–Whitney Students t test with assumed heteroscedasticity (non‐parametric).Statistical analyses were performed using GraphPad Prism  and Excel . All statistical tests used a cutoff 2.4cBioPortal.org datasets were used for the cancer genome atlas (TCGA) bioinformatics analysis. Prostate cancer data sets were selected for the adenocarcinoma study comprised in the firehose legacy data set.cbioportal.org was used as a preferred curated resource of the TCGA datasets for robust harmonized uniformed analyses of prostate cancers.33.1With the availability of diverse molecular genomic data available to appreciate the nature or molecular players in prostate cancer, we accessed TCGA via the cBioPortal. The BMP pathway is a large collection of secreted ligand, soluble antagonists and receptor signaling partners, which we simplified into these three respective categories—ligands, antagonists, and receptor/mediators Figure A. We uti3.250 values, which indicate a therapeutic agent's half‐maximal inhibitory concentration to reduce Cell Titer Glo cell viability. Within the CCLE, we selected the lytic‐like cell lines PC3, 22Rv1, and Du145 in one group and the blastic‐like cell lines LNCaP, VCaP, and MDA‐PCa‐2b in another group to compare BMP signaling components , FN1, and CTSK. C4‐2b cells also resulted in reduced transcription of IGF1R as well as SOX9, while a transcriptional increase of BMP6 was observed when BMP was inhibited with DMH1 , lymph node (163 unique patients) and liver (63 unique patients) groups, which were non‐overlapping to determine first if their mutational landscapes were distinct. We found that the most commonly mutated genes were similar across the three sites such as p53 and AR Figure . Gene ex4When Dr Marshall Urist first discovered the soluble factors to be known as BMPs, it set in motion a fundamental quest for secreted growth factors capable of determining cell fates.Prostate cancer's osteo‐tropism is unique for its predilection to the blastic phenotype whereby bone is not lost from a vicious cycle of osteoclast bone destruction but instead promotes a poorly organized woven matrix of mineralized collagen. BMP driving osteogenesis has been proposed to drive this blastic phenotype and BMP inhibition was hypothesized to reduce the osteogenesis program Figure . In threThere remain unresolved mechanisms for the formation of the blastic lesion or the maintenance of the mixed lesion containing both the blastic scleroses and evidence of heightened resorption in lytic lesions. We were surprised by the lack of transcriptional osteogenesis in blastic‐like cell lines treated with BMP inhibition, thus we turned to compare lytic‐like cell lines for osteogenesis under BMP inhibition Figure . BMPs haMetastatic prostate cancer has been a pioneer for metastatic disease research as numerous patients and their progression have provided scientists with unique datasets for in silico analysis such as the TCGA datasets.5A major limitation of this study is the non‐cell autonomous functions of BMPs. BMPs are used in tissues to coordinate diverse cell behavior and these studies examine BMP inhibition of human prostate cancer cells grown on tissue culture plastic. Additionally the observations made by BMP mRNA in TCGA data sets are not fully representative of BMP signaling via proteomic and direct signaling observations. These TCGA data sets are useful in that they are one of the only that show distinct metastatic sites but unfortunately are not annotated with the clinical diagnosis of bone disease being osteoblastic or osteolytic. Prostate cancer cell lines are limited to less than a dozen models, which may not faithfully represent the full spectrum of disease or include a diverse representation of the many men who suffer from the disease. While the generation of new human and mouse models of prostate cancer continues, these resources should be expanded to further enhance the representation of the metastatic disease and its diverse disease progression in response to frontline therapies to improve future research studies.Meredith D. Provera: Conceptualization (lead); data curation (lead); formal analysis ; methodology ; writing – original draft ; writing – review and editing . Desiree M. Straign: Data curation ; methodology ; supervision ; validation ; writing – review and editing . Parvanee Karimpour: Data curation ; investigation ; writing – original draft . Claire L. Ihle: Conceptualization ; formal analysis ; investigation ; writing – original draft ; writing – review and editing . Philip Owens: Conceptualization ; data curation ; formal analysis ; funding acquisition (lead); investigation ; methodology ; project administration (lead); writing – original draft ; writing – review and editing (lead).The authors have stated explicitly that there are no conflicts of interest in connection with this article.All research was exempt from IRB as all human subjects were not identified and data was from publically available repostitories.FIGURE S1 The BMP pathway regulators and effectors are differentially active in prostate cancer metastatic adenocarcinomas and primary adenocarcinomas. Metastatic (444 patients) and primary tumors (501 patients) from prostate are profiled in two separate studies available at the TCGA and accessed through the cBIO.org portal. Oncoprint graphs indicate whether a specific gene is amplified (red) or increased (pink) within the study or deleted (blue) or decreased (light blue). The percent of patients with any modification is indicated to the left of the graph following the gene name. (A) Soluble BMP ligand antagonist expression in (B) Adenocarcinomas compared with (A) metastases. (C‐D) BMP signaling is mediated by receptors, SMAD intracellular proteins and canonically activate transcription of ID1, ID3 and GATA3 expression in metastatic and primary prostate tumors. Primary tumors are derived from the TCGA legacy firehouse. Genomic profiles of mutations, copy number and mRNA expression relative to diploid samples RNA seq with a z‐score threshold of 2.0. The SU2C metastatic data set allows for the additional tracks of neuroendocrine features as well as tissue specific sites of metastases for comparative analyses.FIGURE S2. Canonical BMP signaling inhibition by DMH1. Prostate cancer cell lines were treated with BMPR1a inhibitor DMH1 for 24 hours at 10 μM concentration. Gene expression for BMP signaling read outs, ID1, SMAD6 and SMAD7 for (A) Blastic‐like and (B) Lytic‐like cell lines.FIGURE S3. The effect of BMP inhibition on human blastic‐like prostate cancer cell lines osteogenesis transcripts. Blastic‐like prostate cancer cell lines were treated with BMP type I receptor inhibitor DMH1 at 10uM for 24 hours. RNA was isolated and an RT2 Profiler PCR Array Human Osteogenesis was run for three prostate cancer lines. The DMH1 sample was compared to an untreated sample (DMSO treated). (A) Genes were increased or decreased 2‐fold in C4 2b cell line. (B) Genes that were increased or decreased by 2‐fold in the VCaP cell line are shown as compared to the untreated group. (C) Genes were increased or decreased 2‐fold in MDA PCA 2B cell line.FIGURE S4. The effect of BMP inhibition on human lytic‐like prostate cancer cell lines osteogenesis transcripts. Lytic‐like prostate cancer cell lines were treated with BMPR1a inhibitor DMH1 for 24 hours. RNA was isolated and an RT2 Profiler PCR Array Human Osteogenesis was run for three prostate cancer lines. The DMH1 sample was compared to an untreated sample. (A) Genes were increased or decreased 2‐fold in PC3 cell line. (B) Genes that were increased or decreased by 2‐fold in the 22Rv1 cell line are shown as compared to the untreated group. (C) Genes were increased or decreased 2‐fold in Du145 cell line.FIGURE S5. Distinct copy number alterations in metastatic prostate cancer by metastasis location. The cBIO portal was used to compare the 2019 PNAS metastatic prostate cancer data set by tissue site. All patients were mutually exclusive and not represented in more than one group.FIGURE S6. BMP Pathway genes. (A) The BMP pathway can be simplified into ligands, soluble antagonists and intracellular signaling components. (B) SYBR qPCR primers used at 60° to validate QIAGEN Osteogenesis qPCR arrays.Click here for additional data file.