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.


STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL. Hodgkin lymphoma (HL) is a germinal-center-derived B-cell malignancy, although rare cases with T-cell origin have been described . The typHRS and LP cells lack typical B-cell characteristics including expression of B-cell-associated developmental transcription factors (TFs). Even the absence of master TFs PAX5 and EBF1 have been reported; however, their reactivation was insufficient to reconstitute the B-cell phenotype ,15,16. THere, we analyzed the EBV-positive HL cell line AM-HLH . InteresCell lines were obtained from the DSMZ : AM-HLH (ACC 908), HDLM-2 (ACC 17), KM-H2 (ACC 8), L-428 (ACC 197), L-540 (ACC 72), L-1236 ACC 530), SUP-HD1 (ACC 574), U-HO1 (ACC 626). Cell culture conditions, culture media and other relevant information on each cell line are described elsewhere , SUP-HD1. Analyze6 cells by electroporation using the EPI-2500 impulse generator  at 350 V for 10 ms. Transfected cells were harvested after 20 h cultivation. Cell stimulations with 20 ng/mL interleukin (IL)6 or IL27  were performed for 4 h, and for 20 h with 10 µg/mL histone deacetylase inhibitor TSA . All knockdown and stimulation experiments were performed twice generating similar results.Gene activities were suppressed via siRNA-mediated knockdown. Gene-specific siRNA oligonucleotides and AllStars negative control siRNA (siCTR) were purchased from Qiagen . A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene . SiRNAs (80 pmol) and plasmid DNA (2 µg) were transfected into 1 × 10For functional testing, treated cells were analyzed with the IncuCyte S3 Live-Cell Analysis System . Apoptotic cells were quantified using the IncuCyte Caspase-3/7 Green Apoptosis Assay diluted at 1:2000 . Live-cell imaging experiments were performed twice with four-fold parallel tests.Total RNA was extracted from cell line samples using TRIzol reagent . cDNA was synthesized by random priming from 5 µg RNA using Superscript II . Expression analysis of EBV-encoded genes LMP1, LMP2A, EBNA1, EBNA2, EBNA3A, EBNA3C and of control gene YY1 was performed as described previously . Reverset-test and the derived p-values are indicated by asterisks .Real-time quantitative (RQ)-PCR analysis was performed with the 7500 Real-time System using commercial buffer and primer sets . For normalization of expression levels, we analyzed the transcript of TATA box binding protein (TBP). We used the ddCT-method, and the obtained values are indicated as fold expression in relation to one selected sample which was set to 1. Quantitative PCR analyses were performed in triplicate. Standard deviations are calculated for each experiment and presented in the figures as error bars. Statistical significance was assessed by Western blots were generated using the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in phosphate-buffered saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), STAT3 , phospho-(P)-STAT3 , and IRF4 (Origene). For loading controls, blots were reversibly stained with Poinceau (Sigma), and the detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies  were linked to peroxidase for detection by Western-Lightning-ECL . Documentation was recorded using the digital system ChemoStar Imager .Immuno-cytology was performed as follows: cells were spun onto slides, air-dried and fixed with methanol/acetic acid for 90 s. The STAT3 antibody (see above) was diluted 1:20 in PBS containing 5% BSA and incubated for 30 min. Washing was performed 3 times with PBS. Preparations were incubated with fluorescent secondary antibody (diluted 1:100) for 20 min. After final washing the cells were mounted in Vectashield , containing DAPI for nuclear staining. Images were captured with an Axion A1 microscope using Axiocam 208 color and software ZEN 3.3 blue edition .For genomic profiling by cytogenetic microarray, the genomic DNA of cell line AM-HLH was prepared by the Qiagen Gentra Puregene Kit (Qiagen). The labelling, hybridization and scanning of HD Cytoscan arrays was performed at the Genome Analytics Facility, Helmholtz Centre for Infection Research , using HD arrays according to the manufacturer’s protocols . Data were visualized and interpreted using the Chromosome Analysis Suite, software version 2.0.1.2 (Affymetrix). https://www.ncbi.nlm.nih.gov/gds/, accessed on 1 February 2022) [Gene expression profiling data from cell lines were generated at the Genome Analytics Facility using HG U133 Plus 2.0 gene chips , as reported previously . Expressry 2022) . Transcrry 2022) .In HL, DLBCL and ALCL, STAT3 aberrantly activates NKL homeobox gene HLX which plays an oncogenic role in these lymphoid malignancies ,19,20. AHowever, RQ-PCR analysis of NKL homeobox gene HLX in cell lines derived from HL, DLBCL and ALCL detected only weak expression levels in AM-HLH. This result contrasted to EBV-negative HL cell line L-540, EBV-positive DLBCL cell line DOHH-2 and all examined EBV-negative ALCL cell lines C. HLX isRQ-PCR analysis of STAT3 in eight HL cell lines showed low expression levels in AM-HLH while HDLM-2, L-428 and L-540 expressed the highest levels A. AccordHowever, the treatment of AM-HLH with IL6 for 4 h resulted in STAT3 phosphorylation and translocation from the cytoplasm into the nucleus C, demonsThe treatment of L-540 cells with the deacetylase inhibitor TSA resulted in the translocation of STAT3 from the nucleus into the cytoplasm D. AccordGenomic aberrations play a basic role in the pathogenesis of HL. Accordingly, the tumor cells contain several chromosomal rearrangements and copy number alterations as described in patients and cell lines ,11,12,13We focused on seven amplicons exhibiting at least a 10-fold rise in their copy numbers which were located at 6p25, 9p24, 9p13, 16p13, 16p12, 16p11 and 20q13 . Gene exIRF4 encodes a hematopoietic TF and was amplified at 6p25 and overexpressed in AM-HLH as confirmed by RQ-PCR and Western blot analysis A. IRF4 hBAG1 encodes an activator of survival factor BCL2 . BAG1 waThe amplifications at 16p harbor several overexpressed genes which, however, indicates no connection to STAT3 or EBV C. In conTo validate these findings and to identify additional genes differentially expressed in AM-HLH, we performed genome-wide comparative gene expression profiling analysis, analyzing the top-1000 up/downregulated genes . AccordiOur approach analyzing genomic and gene expression profiling data from AM-HLH and HL control cell lines revealed interesting candidates potentially involved in STAT3 regulation or lymphomagenesis. These genes and their downstream effects were analyzed by the modulation of their activity. SiRNA-mediated knockdown of IRF4 was confirmed by RQ-PCR and Western blot analysis A. The daBoth, IL6- and IL27-signalling activates STAT3 ,22. AccoKMT2A is a direct target of amplified Zinc-finger TF ZFP64 . KMT2A iTaken together, these data showed that overexpressed IRF4 and NFATC act as STAT3 inhibitors. Nevertheless, STAT3 was still ready for reactivation as demonstrated by treatment with IL6 or IL27. Furthermore, we detected downregulation of STAT3-activators NFIB and HIPK2 and of STAT3-target genes HLX, MIR155 and PAX5. CELF2 is a reported inhibitor of MIR155 and was activated by overexpressed NFATC and KMT2A.We identified IRF4 as an amplified and overexpressed STAT3-inhibitor in AM-HLH. To examine its potential oncogenic activity in AM-HLH we performed live-cell-imaging after siRNA-mediated knockdown of IRF4. The results indicated that IRF4 supported proliferation A and inhThe results of this study are summarized in IRF4 played a central role in STAT3 inhibition. Its gene was amplified at 6p25 and consequently overexpressed. IRF4 also inhibited the expression of NFIB which has been shown to activate STAT3 . FunctioJAK2 represents a major STAT3-activator and was downregulated in AM-HLH although located in an amplicon. This observation indicated that this cell line escapes STAT3-activation. As compared to HLX-positive cell line L-540, HLX target gene SPIB was elevated in AM-HLH. SPIB plays an important role for both, B-cell differentiation and EBV activity, thus, indicating a conflict of two pathognomonic mechanisms ,49,50. PAdditional deregulated players identified in AM-HLH included ZFP64, KMT2A, CELF2, and MIR155. ZFP64 was amplified at 20q13 and overexpressed. This TF has been described as potent activator of KMT2A transcription , which wUsing cell line AM-HLH as a model for EBV-positive HL, we recognized downregulated STAT3 activity. Amplified and overexpressed IRF4 played a major role in STAT3 inhibition. Genes located downstream of STAT3 have been described as both oncogenes and regulators of EBV. STAT3 was still ready for reactivation via IL6- and IL27-signalling, indicating a possible switch in EBV activity. Thus, our study revealed conflicting roles of STAT3 in HL and showed that AM-HLH represents a unique cell line model to gauge therapeutic options for EBV-positive HL patients.


TP53 (p53) is mutated in 80–90% of cases of triple-negative breast cancer (TNBC). Statins, which are widely used to treat elevated cholesterol, have recently been shown to degrade mutant p53 protein and exhibit anti-cancer activity. The aim of this work was to evaluate the potential of statins in the treatment of TNBC. The anti-proliferative effects of 2 widely used statins were investigated on a panel of 15 cell lines representing the different molecular subtypes of breast cancer. Significantly lower IC50 values were found in triple-negative (TN) than in non-TN cell lines  indicating greater sensitivity. Furthermore, cell lines containing mutant p53 were more responsive to both statins than cell lines expressing wild-type p53, suggesting that the mutational status of p53 is a potential predictive biomarker for statin response. In addition to inhibiting proliferation, simvastatin was also found to promote cell cycle arrest and induce apoptosis. Using an apoptosis array capable of detecting 43 apoptosis-associated proteins, a novel protein shown to be upregulated by simvastatin was the IGF-signalling modulator, IGBP4, a finding we confirmed by Western blotting. Finally, we found synergistic growth inhibition between simvastatin and the IGF-1R inhibitor, OSI-906 as well as between simvastatin and doxorubicin or docetaxel. Our work suggests repurposing of statins for clinical trials in patients with TNBC. Based on our findings, we suggest that these trials investigate statins in combination with either doxorubicin or docetaxel and include p53 mutational status as a potential predictive biomarker. Patients with triple-negative breast cancer (TNBC) lack estrogen receptors, progesterone receptors as well as HER2 gene amplification/overexpression. Consequently, these patients cannot be treated with 2 of the most effective therapies currently available for breast cancer, i.e. endocrine and anti-HER2 therapy. Indeed, until recently, the only form of systemic therapy available for patients with TNBC was cytotoxic chemotherapy . RecentlOne of the most successful forms of anti-cancer treatment introduced in recent years is the use of drugs for targeting cancer driver genes. The most frequently occurring driver gene in TNBC is mutations in p53 which are present in 80–90% of these patients , 5. ThusHistorically, however, mutant p53 has proved difficult to target and was thus frequently regarded as “undruggable” , 7. ThisWhile mutant p53-reactivating compounds have been widely investigated , less woIn addition to HSP inhibitors, a group of commonly used drugs known as statins, have also been shown to degrade mutant p53 but not WT p53 , 16. StaBased on the high prevalence of p53 mutations in TNBC, the ability of statins to degrade mutant p53, and thereby inhibit tumour cell proliferation, the aim of our study was to test the hypothesis that these drugs might provide a new treatment for patients with TNBC.All cell lines were originally sourced from the American Type Culture Collection (ATCC), apart from Hs578Ts(i8) which was supplied by Dr. Susan McDonnell, University College, Dublin. All cell lines were maintained in RPMI, supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. All cell culture reagents were purchased from Biosciences  and Sigma-Aldrich . Cells were maintained at 37 °C in a 5% CO2 environment. The p53 mutation status of these cell lines is shown in Table Cell response to statins was assessed by seeding 96-well plates with 5,000 cells per well and allowing to adhere overnight. The following day, fresh media were added, containing statins at the indicated concentration or DMSO. Cells were incubated for 5 days, at which point viability was assessed by adding 3--2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) to 10% of media volume and incubating for 2 h. Absorbance was measured at 570 nm. In some experiments, mevalonate was added at a concentration of 100 or 200 µM.Cells treated with statins for 24 h were harvested and fixed by resuspending in 500 µL PBS followed by slowly adding 4.5 mL ice-cold 70% ethanol. Cells were prepared for cell cycle analysis using FxCycle™ PI/RNase Staining Solution (Thermo-Fisher), following manufacturer’s protocol. Fluorescence was measured on a BD FACSCanto II, using a 488 nm excitation source and a 576 nm emission filter. Cell cycle percentages were calculated using Flowjo software.Cells treated with statins for 48 h were harvested and prepared for analysis using the FITC Annexin V Apoptosis Detection Kit with 7-AAD  following manufacturer’s guidelines. Fluorescence intensity of FITC and 7-AAD was measured on a BD FACSCanto II, using a 488 nm excitation source and a 525 nm or 695 nm emission filter, respectively.Expression of apoptotic proteins was analysed using the Raybiotech Human Apoptosis Array following manufacturer’s guidelines. Briefly, protein was isolated from SUM159 cells following treatment for 24 h with 10 µM simvastatin or vehicle control, using the provided lysis buffer. Membranes were blocked and incubated with protein samples overnight at 4 °C. After washing with the provided buffers, membranes were incubated overnight again at 4 °C with detection antibody. Membranes were incubated at room temperature with HRP-Streptavidin for 2 h. Following a final wash stage, membranes were incubated with detection buffer and imaged using a Licor C-DiGit Blot Scanner. ImageJ software was used to generate densitometry values which were then normalised to the internal controls on the membrane.Protein was isolated and prepared for analysis by Western blotting as previously described . PrimaryEfficacy of drug combinations was performed following the same protocol as proliferation assays. Drugs were tested as both single agents and in combination. Combination index (CI) values were calculated using Compusyn software . CI valuAll experiments were repeated at least 3 times. All statistical analysis was performed using GraphPad Prism 8 software. Significance was determined using Student t tests or ANOVA post-hoc analysis. A p value of less than 0.05 was deemed statistically significant.r = 0.94, p < 0.0001)   , HER2 (2 cell lines) and TNBC (9 cell lines). Both statins reduced cell viability in a dose-dependent and cell type-dependent manner , it also reduces levels of the intermediate product, MVA . To confAs simvastatin was the more potent of the two statins investigated, we focussed on it for the rest of our study. To investigate if simvastatin could induce cell cycle arrest in TNBC cells, we measured DNA content by propidium iodide staining and flow-cytometry in 4 TNBC cell lines following 24 h treatment. Representative graphs of simvastatin-treated MDA-MB-231 cells are shown below  and 46% (p < 0.001) cells staining positive for annexin-V, respectively. Similar levels of annexin-V positivity were seen in Hs578T  annexin-V staining . The combination was also effective in SUM159 cells .Finally, we tested the combination of simvastatin and OSI-906 on apoptosis. MDA-MB-231 and SUM159 cells were treated with a low dose of simvastatin (1 µM) that, by itself, did not significantly induce apoptosis in either cell line Fig. A, B. SimAs statins are unlikely to be used alone in cancer treatment, we also tested the anti-proliferative effect of combined treatment with a statin and standard chemotherapy drugs used for the treatment of breast cancer. As shown in Table Here, we show that although 2 widely used statins decreased the proliferation of breast cancer cell lines representing the main molecular subtypes of breast cancer, their inhibitory impact was significantly more potent in TNBC than in non-TNBC cell lines. A possible explanation for the enhanced sensitivity of the TNBC versus the non-TNBC cell lines may relate to the higher prevalence of p53 mutations in this breast cancer subtype. Indeed, as mentioned in the Introduction above, our findings are consistent with previous observations in which treatment with statins was reported to result in the degradation of mutant but not wild-type p53 , 16. PrePreviously, Parrales et al. reported that lovastatin degraded p53 with conformational mutations but had minimal effects on contact mutations . Based oAlthough our results show that two different statins reduced the proliferation of TNBC cell lines, these drugs are unlikely to be used alone for the treatment of this form of breast cancer. We therefore evaluated the effects of combining simvastatin with two frequently used drugs to treat TNBC, i.e. docetaxel and doxorubicin. Both these cytotoxic drugs in combination with simvastatin synergistically enhanced growth inhibition in the 2 TNBC cell lines investigated, suggesting that combined treatment with simvastatin and docetaxel or doxorubicin might be investigated in a clinical trial.A novel finding in this report was that simvastatin upregulated levels of IGBPF4. Although we did not investigate the mechanism of this upregulation or its possible implication for statin actions, previous studies showed that IGFBP4 binds and sequesters IGF-1 ligands, thus acting to dampen IGFR1 signalling . ConsistWe should state however, that there are several other mechanisms other than via upregulation of IGBP4 by which statins could decrease cell proliferation or promote apoptosis. These include a reduction in different cell signalling systems such as from RAS, RHO, Hedgehog, YAP or TAZ . Furthep = 0.01), longer breast cancer-free interval  and longer distant recurrence-free interval  [Consistent with our preclinical studies, several clinical studies have shown that the use of statins was associated with improved outcome in breast cancer patients. Thus following a systematic review of the literature and meta-analysis, Manthravadi et al.  identifi = 0.03) .p = 0.022) and overall survival  in patients with TNBC (n = 1534). In contrast, there was no association with breast cancer-specific survival  or overall survival  in those without TN disease . Since cell lines containing mutant p53 appear to be more sensitive to statins than p53 wild-type cells [Of particular relevance to our study was the recent report showing a significant benefit of statins in patients with TNBC but not in those with non-triple-negative disease . Thus, ipe cells  theoretiDespite the multiplicity of preclinical and epidemiological studies linking statin use with anti-cancer activity across different types of cancer, there are little data from randomized clinical trials that treatment with these drugs improves outcome for patients with cancer. Statins however, are one of the most commonly prescribed classes of drug worldwide, with well-established safety and dosage profiles . Their lIn conclusion, our data described in this article may be informative with respect to the design of clinical trials involving statins for the treatment of breast cancer. We show that the anti-proliferative effect of simvastatin is enhanced by combination with docetaxel or doxorubicin. Furthermore, our preliminary finding when combined with that of others  suggests