Abstract Objective: Charcot‐Marie‐Tooth (CMT) disease comprises a large number of genetically distinct forms of inherited peripheral neuropathies. The relative uniform phenotypes in many patients with CMT make it difficult to decide which of the over 35 known CMT genes are affected in a given patient. Genetic testing decision trees are therefore broadly based on a small number of major subtypes (eg, CMT1, CMT2) and the observed mutation frequency for CMT genes. Since conventional genetic testing is expensive many rare genes are not being tested for at all. Methods: Whole‐exome sequencing has recently been introduced as a novel and alternative approach. This method is capable of resequencing a nearly complete set of coding exons in an individual. We performed whole‐exome sequencing in an undiagnosed family with CMT. Results: Within over 24,000 variants detected in 2 exomes of a CMT family, we identified a nonsynonymous GJB1 ( Cx32 ) mutation. This variant had been reported previously as pathogenic in X‐linked CMT families. Sanger sequencing confirmed complete cosegregation in the family. Affected individuals had a marked early involvement of the upper distal extremities and displayed a mild reduction of nerve conduction velocities. Interpretation: We have shown for the first time in a genetically highly heterogeneous dominant disease that exome sequencing is a valuable method for comprehensive medical diagnosis. Further improvements of exon capture design, next‐generation sequencing accuracy, and a constant price decline will soon lead to the adoption of genomic approaches in gene testing of Mendelian disease. Ann Neurol 2011;
ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I's role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers (N-cadherin, slug, snail, fibronectin) and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers (E-cadherin, occludin) were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I's role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I's tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR. ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I's role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers (N-cadherin, slug, snail, fibronectin) and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers (E-cadherin, occludin) were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I's role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I's tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR. Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a dismal 5-year survival rate of less than 9% (https://www.cancer.org/cancer/pancreatic-cancer/detection-diagnosis-staging/survival-rates.html, accessed September 1, 2020). Most patients are diagnosed after metastatic lesions have formed, making treatment difficult and often unsuccessful. It is thought that PDAC metastasizes early during the carcinogenic process owing to the emergence of stem-like cancer cells with high metastatic potential, termed cancer stem cells (CSCs) (1Rhim A.D. Mirek E.T. Aiello N.M. Maitra A. Bailey J.M. McAllister F. Reichert M. Beatty G.L. Rustgi A.K. Vonderheide R.H. Leach S.D. Stanger B.Z. EMT and dissemination precede pancreatic tumor formation.Cell. 2012; 148: 349-361Abstract Full Text Full Text PDF PubMed Scopus (1282) Google Scholar). CSCs are inherently invasive and apoptosis resistant, and hence, these cells are major drivers of cancer progression (2Shibue T. Weinberg R.A. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications.Nat. Rev. Clin. Oncol. 2017; 14: 611-629Crossref PubMed Scopus (892) Google Scholar). Considerable efforts have been aimed at elucidating the functional role of stem-like cancer cells in metastasis; however, the contribution of the cellular glycome to this process has received insufficient attention. Recent studies have suggested that the glycosyltransferase, ST6Gal-I, promotes CSC characteristics (3Schultz M.J. Holdbrooks A.T. Chakraborty A. Grizzle W.E. Landen C.N. Buchsbaum D.J. Conner M.G. Arend R.C. Yoon K.J. Klug C.A. Bullard D.C. Kesterson R.A. Oliver P.G. O'Connor A.K. Yoder B.K. et al.The tumor-associated glycosyltransferase ST6Gal-I regulates stem cell transcription factors and confers a cancer stem cell phenotype.Cancer Res. 2016; 76: 3978-3988Crossref PubMed Scopus (71) Google Scholar) and acts as a survival factor to protect cells against cytotoxic assaults including chemotherapy (4Chakraborty A. Dorsett K.A. Trummell H.Q. Yang E.S. Oliver P.G. Bonner J.A. Buchsbaum D.J. Bellis S.L. ST6Gal-I sialyltransferase promotes chemoresistance in pancreatic ductal adenocarcinoma by abrogating gemcitabine-mediated DNA damage.J. Biol. Chem. 2018; 293: 984-994Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 5Chen X. Wang L. Zhao Y. Yuan S. Wu Q. Zhu X. Niang B. Wang S. Zhang J. ST6Gal-I modulates docetaxel sensitivity in human hepatocarcinoma cells via the p38 MAPK/caspase pathway.Oncotarget. 2016; 7: 51955-51964Crossref PubMed Scopus (28) Google Scholar, 6Park J.J. Yi J.Y. Jin Y.B. Lee Y.J. Lee J.S. Lee Y.S. Ko Y.G. Lee M. 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ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines.Cancer Res. 2013; 73: 2368-2378Crossref PubMed Scopus (99) Google Scholar, 11Hsieh C.C. Shyr Y.M. Liao W.Y. Chen T.H. Wang S.E. Lu P.C. Lin P.Y. Chen Y.B. Mao W.Y. Han H.Y. Hsiao M. Yang W.B. Li W.S. Sher Y.P. Shen C.N. Elevation of beta-galactoside alpha2,6-sialyltransferase 1 in a fructoseresponsive manner promotes pancreatic cancer metastasis.Oncotarget. 2017; 8: 7691-7709Crossref PubMed Scopus (28) Google Scholar, 12Lise M. Belluco C. Perera S.P. Patel R. Thomas P. Ganguly A. Clinical correlations of alpha2,6-sialyltransferase expression in colorectal cancer patients.Hybridoma. 2000; 19: 281-286Crossref PubMed Scopus (57) Google Scholar, 13Wichert B. Milde-Langosch K. Galatenko V. Schmalfeldt B. Oliveira-Ferrer L. Prognostic role of the sialyltransferase ST6GAL1 in ovarian cancer.Glycobiology. 2018; 28: 898-903Crossref PubMed Scopus (16) Google Scholar), and high expression of this enzyme correlates with a poor patient prognosis (3Schultz M.J. Holdbrooks A.T. Chakraborty A. Grizzle W.E. Landen C.N. Buchsbaum D.J. Conner M.G. Arend R.C. Yoon K.J. Klug C.A. Bullard D.C. Kesterson R.A. Oliver P.G. O'Connor A.K. Yoder B.K. et al.The tumor-associated glycosyltransferase ST6Gal-I regulates stem cell transcription factors and confers a cancer stem cell phenotype.Cancer Res. 2016; 76: 3978-3988Crossref PubMed Scopus (71) Google Scholar, 11Hsieh C.C. Shyr Y.M. Liao W.Y. Chen T.H. Wang S.E. Lu P.C. Lin P.Y. Chen Y.B. Mao W.Y. Han H.Y. Hsiao M. Yang W.B. Li W.S. Sher Y.P. Shen C.N. Elevation of beta-galactoside alpha2,6-sialyltransferase 1 in a fructoseresponsive manner promotes pancreatic cancer metastasis.Oncotarget. 2017; 8: 7691-7709Crossref PubMed Scopus (28) Google Scholar, 12Lise M. Belluco C. Perera S.P. Patel R. Thomas P. Ganguly A. Clinical correlations of alpha2,6-sialyltransferase expression in colorectal cancer patients.Hybridoma. 2000; 19: 281-286Crossref PubMed Scopus (57) Google Scholar, 13Wichert B. Milde-Langosch K. Galatenko V. Schmalfeldt B. Oliveira-Ferrer L. Prognostic role of the sialyltransferase ST6GAL1 in ovarian cancer.Glycobiology. 2018; 28: 898-903Crossref PubMed Scopus (16) Google Scholar). Interestingly, ST6Gal-I expression is induced by oncogenic Ras signaling (14Dalziel M. Dall'Olio F. Mungul A. Piller V. Piller F. Ras oncogene induces beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter.Eur. J. Biochem. 2004; 271: 3623-3634Crossref PubMed Scopus (41) Google Scholar). Activating mutations in K-Ras are found in more than 90% of patients with PDAC (15Kiaris H. Spandidos D. Mutations of ras genes in human tumors (review).Int. J. Oncol. 1995; 7: 413-421PubMed Google Scholar), and these mutant isoforms appear early in tumor development, as indicated by their presence in the premalignant lesions, PanINs (16Kim J.Y. Hong S.M. Precursor lesions of pancreatic cancer.Oncol. Res. Treat. 2018; 41: 603-610Crossref PubMed Scopus (4) Google Scholar). Likewise, ST6Gal-I is strongly expressed in PanINs, whereas normal pancreatic acinar cells lack detectable ST6Gal-I protein expression (3Schultz M.J. Holdbrooks A.T. Chakraborty A. Grizzle W.E. Landen C.N. Buchsbaum D.J. Conner M.G. Arend R.C. Yoon K.J. Klug C.A. Bullard D.C. Kesterson R.A. Oliver P.G. O'Connor A.K. Yoder B.K. et al.The tumor-associated glycosyltransferase ST6Gal-I regulates stem cell transcription factors and confers a cancer stem cell phenotype.Cancer Res. 2016; 76: 3978-3988Crossref PubMed Scopus (71) Google Scholar). An enrichment in tumor cell sialylation has long been implicated in neoplastic transformation and tumor-promoting cellular behaviors such as invasiveness and apoptosis resistance (17Schultz M.J. Swindall A.F. Bellis S.L. Regulation of the metastatic cell phenotype by sialylated glycans.Cancer Metastasis Rev. 2012; 31: 501-518Crossref PubMed Scopus (186) Google Scholar, 18Harduin-Lepers A. Krzewinski-Recchi M.A. Colomb F. Foulquier F. Groux-Degroote S. Delannoy P. Sialyltransferases functions in cancers.Front Biosci. (Elite Ed. 2012; 4: 499-515Crossref PubMed Google Scholar). The addition of sialic acid to membrane receptors can profoundly affect cell signaling and phenotype owing to sialylation-dependent changes in receptor features such as conformation, oligomerization, and/or cell surface retention. In particular, the α2-6 sialic acid linkage is often increased upon malignant transformation (19Dall'Olio F. Malagolini N. Trinchera M. Chiricolo M. Mechanisms of cancer-associated glycosylation changes.Front Biosci. 2012; 17: 670-699Crossref PubMed Scopus (107) Google Scholar) and is similarly elevated in certain nonmalignant stem cell populations (20Hasehira K. Tateno H. Onuma Y. Ito Y. Asashima M. Hirabayashi J. Structural and quantitative evidence for dynamic glycome shift on production of induced pluripotent stem cells.Mol. Cell Proteomics. 2012; 11: 1913-1923Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). As ST6Gal-I is the predominant enzyme responsible for the α2-6 sialylation of N-glycosylated proteins, understanding its function in cancer is critical. ST6Gal-I imparts a malignant cell phenotype by regulating, via sialylation, key receptors that control tumor-associated signaling networks. For example, ST6Gal-I-mediated sialylation of the β1 integrin promotes cell migration and invasion (21Seales E.C. Jurado G.A. Brunson B.A. Wakefield J.K. Frost A.R. Bellis S.L. Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility.Cancer Res. 2005; 65: 4645-4652Crossref PubMed Scopus (241) Google Scholar, 22Zhu Y. Srivatana U. Ullah A. Gagneja H. Berenson C.S. Lance P. Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro.Biochim. Biophys. Acta. 2001; 1536: 148-160Crossref PubMed Scopus (84) Google Scholar), whereas α2-6 sialylation of the Fas and tumor necrosis factor receptor 1 death receptors inhibits apoptosis by hindering receptor internalization (23Swindall A.F. Bellis S.L. Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells.J. Biol. Chem. 2011; 286: 22982-22990Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 24Holdbrooks A.T. Britain C.M. Bellis S.L. ST6Gal-I sialyltransferase promotes tumor necrosis factor (TNF)-mediated cancer cell survival via sialylation of the TNF receptor 1 (TNFR1) death receptor.J. Biol. Chem. 2018; 293: 1610-1622Abstract Full Text Full Text PDF PubMed Google Scholar). Furthermore, our group recently determined that sialylation of epidermal growth factor receptor (EGFR) by ST6Gal-I enhances both basal and ligand-dependent EGFR activation and protects against gefitinib-induced apoptosis (25Britain C.M. Holdbrooks A.T. Anderson J.C. Willey C.D. Bellis S.L. Sialylation of EGFR by the ST6Gal-I sialyltransferase promotes EGFR activation and resistance to gefitinib-mediated cell death.J. Ovarian Res. 2018; 11: 12Crossref PubMed Scopus (37) Google Scholar). EGFR is a receptor tyrosine kinase with a rich history in cancer pathogenesis. EGFR is heavily glycosylated, and it is well known that the N-glycans play a major part in modulating EGFR structure and function (26Whitson K.B. Whitson S.R. Red-Brewer M.L. McCoy A.J. Vitali A.A. Walker F. Johns T.G. Beth A.H. Staros J.V. Functional effects of glycosylation at Asn-579 of the epidermal growth factor receptor.Biochemistry. 2005; 44: 14920-14931Crossref PubMed Scopus (44) Google Scholar, 27Azimzadeh Irani M. Kannan S. Verma C. Role of N-glycosylation in EGFR ectodomain ligand binding.Proteins. 2017; 85: 1529-1549Crossref PubMed Scopus (21) Google Scholar, 28Tsuda T. Ikeda Y. Taniguchi N. The Asn-420-linked sugar chain in human epidermal growth factor receptor suppresses ligand-independent spontaneous oligomerization. Possible role of a specific sugar chain in controllable receptor activation.J. Biol. Chem. 2000; 275: 21988-21994Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The sialylation of EGFR can influence ligand binding, receptor clustering, and, consequently, downstream signaling (29Yen H.Y. Liu Y.C. Chen N.Y. Tsai C.F. Wang Y.T. Chen Y.J. Hsu T.L. Yang P.C. Wong C.H. Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 6955-6960Crossref PubMed Scopus (61) Google Scholar, 30Mathew M.P. Tan E. Saeui C.T. Bovonratwet P. Sklar S. Bhattacharya R. Yarema K.J. Metabolic flux-driven sialylation alters internalization, recycling, and drug sensitivity of the epidermal growth factor receptor (EGFR) in SW1990 pancreatic cancer cells.Oncotarget. 2016; 7: 66491-66511Crossref PubMed Scopus (18) Google Scholar). The activation of EGFR elicits a variety of biological outcomes, including cell proliferation, the cell's response to DNA damage, and the epithelial to mesenchymal transition (EMT) (31Chen D.J. Nirodi C.S. The epidermal growth factor receptor: a role in repair of radiation-induced DNA damage.Clin. Cancer Res. 2007; 13: 6555-6560Crossref PubMed Scopus (147) Google Scholar, 32Voon D.C. Wang H. Koo J.K. Chai J.H. Hor Y.T. Tan T.Z. Chu Y.S. Mori S. Ito Y. EMT-induced stemness and tumorigenicity are fueled by the EGFR/Ras pathway.PLoS One. 2013; 8: e70427Crossref PubMed Scopus (47) Google Scholar, 33Zuo J.H. Zhu W. Li M.Y. Li X.H. Yi H. Zeng G.Q. Wan X.X. He Q.Y. Li J.H. Qu J.Q. Chen Y. Xiao Z.Q. Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin.J. Cell Biochem. 2011; 112: 2508-2517Crossref PubMed Scopus (190) Google Scholar). As with CSCs, cancer cells undergoing EMT reactivate developmental pathways that facilitate invasiveness and apoptosis resistance. In fact, EMT has been proposed as a major mechanism responsible for generating CSCs (34Ye X. Weinberg R.A. Epithelial-mesenchymal plasticity: a central regulator of cancer progression.Trends Cell Biol. 2015; 25: 675-686Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). A role for ST6Gal-I in TGFβ-driven EMT has been previously reported. Gu's group found that ST6Gal-I was selectively upregulated by TGFβ in the GE11 mouse epithelial cell model, and importantly, ST6Gal-I activity was required for TGFβ-stimulated EMT (35Lu J. Isaji T. Im S. Fukuda T. Hashii N. Takakura D. Kawasaki N. Gu J. beta-Galactoside alpha2,6-sialyltranferase 1 promotes transforming growth factor-beta-mediated epithelial-mesenchymal transition.J. Biol. Chem. 2014; 289: 34627-34641Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, the contribution of ST6Gal-I to EGFR-mediated EMT has not previously been investigated. Given the relationship between EGFR and EMT, combined with the finding that ST6Gal-I enhances EGFR activation, we interrogated whether α2-6 sialylation of EGFR promotes EMT in PDAC cells. The Suit2 PDAC cell line and its isogenic, metastatic subclones, S2-013 and S2-LM7AA, were used to delineate the role of ST6Gal-I in EMT. Notably, ST6Gal-I is markedly upregulated in the metastatic subclones as compared with parental Suit2 cells. RNA-Seq experiments highlighted EMT, cell motility, and stem cell–associated gene networks as prominent pathways upregulated in the metastatic lines. Supporting a functional role for ST6Gal-I in the metastatic phenotype, forced expression of ST6Gal-I in the parental, poorly metastatic Suit2 line (with low endogenous ST6Gal-I) conferred a phenotype similar to that of the metastatic subclones, evidenced by enrichment in EMT, stem cell, and cell motility networks. To further establish a role for ST6Gal-I in EMT, ST6Gal-I expression was knocked down in the metastatic lines, complementing the overexpression of ST6Gal-I in the parental line. In all three of the cell models, high expression of ST6Gal-I led to increased α2-6 sialylation and activation of EGFR, as well as upregulated expression of mesenchymal markers and cell invasiveness. Moreover, the EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR sialylation and activation, EMT marker expression, and invasiveness in Suit2 and S2-LM7AA cells. In the aggregate, these results highlight the importance of ST6Gal-I activity in driving EMT, a critical process that promotes metastatic disease. Immunoblotting for ST6Gal-I in multiple human PDAC cell lines revealed that, although most lines had considerable ST6Gal-I expression, the Suit2 line had negligible levels of ST6Gal-I (Fig. 1A). Suit2 cells have relatively low metastatic potential in animal models, and in order to study the metastatic process, other investigators have developed metastatic subclones of the Suit2 line. For example, the metastatic S2-LM7AA line was generated through iterative in vivo selection, yielding a population that reliably metastasizes to the liver following injection into the pancreas (36Kirby M.K. Ramaker R.C. Gertz J. Davis N.S. Johnston B.E. Oliver P.G. Sexton K.C. Greeno E.W. Christein J.D. Heslin M.J. Posey J.A. Grizzle W.E. Vickers S.M. Buchsbaum D.J. Cooper S.J. et al.RNA sequencing of pancreatic adenocarcinoma tumors yields novel expression patterns associated with long-term survival and reveals a role for ANGPTL4.Mol. Oncol. 2016; 10: 1169-1182Crossref PubMed Scopus (30) Google Scholar). A second metastatic subclone, S2-013, metastasizes to the lungs when grown as a subcutaneous tumor (37Iwamura T. Taniguchi S. Kitamura N. Yamanari H. Kojima A. Hidaka K. Setoguchi T. Katsuki T. Correlation between CA19-9 production in vitro and histological grades of differentiation in vivo in clones isolated from a human pancreatic cancer cell line (SUIT-2).J. Gastroenterol. Hepatol. 1992; 7: 512-519Crossref PubMed Scopus (42) Google Scholar, 38Taniguchi S. Iwamura T. Katsuki T. Correlation between spontaneous metastatic potential and type I collagenolytic activity in a human pancreatic cancer cell line (SUIT-2) and sublines.Clin. Exp. Metastasis. 1992; 10: 259-266Crossref PubMed Scopus (60) Google Scholar). Significantly, both of the Suit2-derived metastatic subclones displayed elevated ST6Gal-I expression when compared with the parental Suit2 line (Fig. 1B). RNA-Seq was conducted on the S2-LM7AA and S2-013 subclones to gain insight into metastasis-associated pathways. To determine whether the upregulation of ST6Gal-I in these subclones contributed to metastatic characteristics, RNA-Seq was also performed on parental Suit2 cells with forced ST6Gal-I overexpression (OE) or cells alternatively transduced with an empty vector (EV) construct (Fig. 1C). We first compared the RNA-Seq data generated from the two metastatic subclones. Table S1 shows the top 50 upregulated (A) and downregulated (B) genes in the S2-LM7AA and S2-013 lines, as compared with poorly metastatic Suit2 EV cells. RNA-Seq confirmed a 3.5-fold upregulation of ST6Gal-I in S2-LM7AA cells and 16-fold upregulation in S2-013 cells (not shown). Ingenuity Pathway Analysis (IPA) was employed to identify the top 20 Biological Functions altered in the metastatic lines relative to Suit EV cells. As shown in Figure 1, D and E, a strong correspondence was noted between the two subclones, illustrated by the fact that 17 of 20 of the Biological Functions were shared (denoted by red bars). These included cancer, gastrointestinal disease, developmental pathways (organismal development, tissue development), and cancer-associated cell functions (cellular movement, cellular development, cellular growth and proliferation). Gene Set Enrichment Analysis (GSEA) of the normalized gene expression showed that, compared with Suit2 EV cells, S2-LM7AA and S2-013 cells were enriched in stemness-associated networks (Wnt, Hedgehog), EMT, and processes related to EMT such as hypoxia (Fig. 1, F and G). We also utilized the IPA Upstream Regulator module to determine that the two metastatic subclones exhibited activation of regulators central to stem cell networks, EMT, cell growth and proliferation, and cell migration (Table S2). Given the upregulation of ST6Gal-I observed in the metastatic subclones, we compared RNA-Seq data from ST6Gal-I OE and EV cells to determine whether forced overexpression of ST6Gal-I was sufficient to confer metastatic characteristics. Table S3 shows the top 50 upregulated and downregulated genes in Suit2 OE cells relative to EV cells. An analysis of the top 20 IPA Biological Functions (Fig. 2A) revealed that forced expression of ST6Gal-I in Suit2 cells altered 14 of 17 of the pathways that were modulated in both of the metastatic subclones (red bars). Another two of the Functions were common to one of the two metastatic lines (green bars). Functions shared with both of the metastatic subclones included cancer, gastrointestinal disease, developmental pathways (organismal development, tissue development), and tumorigenic cell functions (cellular movement, cellular development, cellular growth and proliferation). GSEA indicated that ST6Gal-I overexpression activated Wnt, Hedgehog, EMT, and hypoxia networks (Fig. 2B). In addition, the IPA Upstream Regulators activated in Suit2 OE cells were very similar to those that activated in the metastatic subclones, specifically, regulators involved in EMT, stemness, hypoxia, and cell motility (Table S4). To pinpoint pathways that may be particularly important for metastasis, we identified the Upstream Regulators that were coordinately activated in S2-013 and S2-LM7AA cells (Table 1A). We then compared these Upstream Regulators to those altered in Suit2 OE cells (Table 1A). As shown, about two-thirds (41/63) of the pathways activated in both of the metastatic lines were also activated in Suit2 OE cells relative to Suit2 EV cells. These Upstream Regulators included molecules involved in (i) stemness (WNT3A, β-catenin, LEF1, SHH, JAG1, GLI1, WBP2); (ii) EMT (TGFβ1, SMAD1-4, BMP2, GDF9, GLI1, HIF1A, WWTR1, ROR1, MRTFA, JAK1/2); and (iii) cell migration (ROCK1, RAF1, ARNT2, ROR1, SIM1, TEAD4, WWTR1, MRTFA). Finally, we screened for shared pathways in the IPA Biological Functions and Canonical Pathways databases and observed substantial activation of pathways involved in EMT and cell motility in the three lines with high ST6Gal-I expression, S2-013, S2-LM7AA, and Suit2 OE (Table 1B). Taken together, these data strongly suggest that the forced expression of ST6Gal-I in the parental, poorly metastatic Suit2 line is sufficient to activate many of the metastasis-associated processes upregulated in the metastatic subclones, S2-013 and S2-LM7AA.Table 1Pathways Shared by Metastatic Subclones and Suit2 OE cellsA. Upstream regulators with predicted activationRegulatorS2-013 versusS2-LM7AA versusSuit2 OE versusSuit2 EVSuit2 EVSuit2 EVZ scoreZ scoreZ scoreAPLN2.1641.5542.728ARNT23.5692.3354BDNF2.9432.9142.58BMP21.9811.622.381BRD42.2732.1782.626CBX53.3172.414CCR23.6873.1111.89CG1.8572.2682.287CIITA2.271.9461.82CLEC4G1.9942.121CTNNB12.4933.0431.656D-glucose2.5692.8612.118DSCAM4.1222.6832.688DSCAML13.4521.8773.3EREG2.0121.982F22.453.4412.787FBXO322.4671.655GDF91.742.3921.936GLI12.4332.7872.208Growth hormone2.2261.8072.469HIF1A2.6951.7063.905HRG2.1382.2362IL102.1151.674IL61.5052.34JAG12.3152.2233.086JAK1/22.0651.7322.433KDM3A2.1972.241KMT2D2.4681.694LATS22.5341.706LEF12.0832.3143.026LLGL22.8873.3172.646MAPK13.0462.852MRTFA1.6743.4912.078MRTFB3.4664.1822.883MYOD11.9012.166NMNAT12.1971.976NORAD2.1382.7141.974NOTCH12.8421.988NRG13.7293.7893.696NSUN63.52.8872.646PLAG12.3771.912progesterone1.8612.311RAF11.9792.1632.936ROCK12.1782.6212.425ROR12.0742.8132.236RXFP21.6332SHH1.8232.952.689SIM13.6372.0383.615SMAD11.6762.8052.038Smad2/3-Smad41.6981.983.296SMAD32.7962.7912.264SNAI22.3431.651STAT5B2.3361.681TEAD13.9792.309TEAD23.32.53TEAD33.5782.111TEAD44.0862.1372.449Tgf beta3.4524.0163.543TGFB13.1414.5573.943WBP22.4192.2123.036WNT3A1.9543.3883.535WWTR12.6972.0411.698ZEB11.8222.06B. Biological functions and canonical pathwaysRegulatorS2-013 versusS2-LM7AA versusSuit2 OE versusSuit2 EVSuit2 EVSuit2 EVZ scoreZ scoreZ scoreRegulation of EMT by growth factors pathway2.3092.3572.137Regulation of EMT in development pathway2.3332.3572.132Colorectal cancer metastasis signaling2.8372.5562.188Ephrin receptor signaling2.8282.6732.414Integrin signaling3.4641.7892.646Migration of cells4.7863.9723.622Migration of tumor cell lines3.2061.7983.64Microtubule dynamics6.3434.3023.901Organization of cytoskeleton6.0424.2674.084Chemotaxis5.8393.6482.004Formation of cellular protrusions6.1043.6363.881Cell movement5.6294.2193.315Cell movement of tumor cell lines3.5521.5432.709 Open table in a new tab Based on the RNA-Seq results, we further investigated the role of ST6Gal-I in EMT. To supplement the Suit2 OE cell model (Fig. 3A), ST6Gal-I was knocked down (KD) in the metastatic S2-LM7AA and S2-013 lines (Fig. 3, B and C, respectively). A nontargeting shRNA sequence was used as the control (shC). The expression of EMT markers was examined in cells with modulated ST6Gal-I expression. A switch between E-cadherin, an epithelial cadherin, and N-cadherin, a mesenchymal cadherin, is a hallmark of EMT. In addition, cells that have undergone EMT exhibit an upregulation in the mesenchymal transcription factors, slug and snail. As compared with Suit2 EV cells, Suit2 OE cells had increased expression of the mesenchymal markers, snail and N-cadherin, although slug expression was unchanged (Fig. 3A). Contrarily, the epithelial marker, E-cadherin, was reduced in OE cells. Consistent with t
Abstract Breast cancer is a leading cause of cancer-related deaths among women, and current therapies benefit only a subset of these patients. Here, we show that ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in patient-derived breast cancer samples, and UBE2T overexpression predicts poor prognosis. We demonstrate that the transcription factor AP-2 alpha (TFAP2A) is necessary for the overexpression of UBE2T in breast cancer cells, and UBE2T inhibition suppresses breast cancer tumor growth in cell culture and in mice. RNA sequencing analysis identified interferon alpha–inducible protein 6 (IFI6) as a key downstream mediator of UBE2T function in breast cancer cells. Consistently, UBE2T inhibition downregulated IFI6 expression, promoting DNA replication stress, cell cycle arrest, and apoptosis and suppressing breast cancer cell growth. Breast cancer cells with IFI6 inhibition displayed similar phenotypes as those with UBE2T inhibition, and ectopic IFI6 expression in UBE2T-knockdown breast cancer cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Taken together, our study identifies UBE2T as a facilitator of breast cancer tumor growth and provide a rationale for targeting UBE2T for breast cancer therapies.
The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate.
The von Willebrand Factor type A domain is the prototype for a protein superfamily. It possesses no significant sequence similarity to any known protein structure. Secondary structure predictions indicate a largely alternating pattern of six alpha-helices and six beta-strands. A protein fold for this domain is proposed to correspond to a doubly-wound open twisted beta-sheet structure flanked by alpha-helices. Close agreement was found with the GTP-binding domain of human ras-p21, provided that an extra alpha-helix was inserted. The structure of the predicted fold showed high compatibility with the proximate location of two Mg(2+)-binding Asp residues, two disulphide-bridged Cys residues, and other known functional attributes of this domain.
The compact genome of Fugu rubripes has been sequenced to over 95% coverage, and more than 80% of the assembly is in multigene-sized scaffolds. In this 365-megabase vertebrate genome, repetitive DNA accounts for less than one-sixth of the sequence, and gene loci occupy about one-third of the genome. As with the human genome, gene loci are not evenly distributed, but are clustered into sparse and dense regions. Some “giant” genes were observed that had average coding sequence sizes but were spread over genomic lengths significantly larger than those of their human orthologs. Although three-quarters of predicted human proteins have a strong match to Fugu , approximately a quarter of the human proteins had highly diverged from or had no pufferfish homologs, highlighting the extent of protein evolution in the 450 million years since teleosts and mammals diverged. Conserved linkages between Fugu and human genes indicate the preservation of chromosomal segments from the common vertebrate ancestor, but with considerable scrambling of gene order.
Myosin heavy chain genes (MYHs) are the most important functional domains of myosins, which are highly conserved throughout evolution. The human genome contains 15 MYHs, whereas the corresponding number in teleost appears to be much higher. Although teleosts comprise more than one-half of all vertebrate species, our knowledge of MYHs in teleosts is rather limited. A comprehensive analysis of the torafugu (Takifugu rubripes) genome database enabled us to detect at least 28 MYHs, almost twice as many as in humans. RT-PCR revealed that at least 16 torafugu MYH representatives (5 fast skeletal, 3 cardiac, 2 slow skeletal, 1 superfast, 2 smooth, and 3 nonmuscle types) are actually transcribed. Among these, MYH(M743-2) and MYH(M5) of fast and slow skeletal types, respectively, are expressed during development of torafugu embryos. Syntenic analysis reveals that torafugu fast skeletal MYHs are distributed across five genomic regions, three of which form clusters. Interestingly, while human fast skeletal MYHs form one cluster, its syntenic region in torafugu is duplicated, although each locus contains just a single MYH in torafugu. The results of the syntenic analysis were further confirmed by corresponding analysis of MYHs based on databases from Tetraodon, zebrafish, and medaka genomes. Phylogenetic analysis suggests that fast skeletal MYHs evolved independently in teleosts and tetrapods after fast skeletal MYHs had diverged from four ancestral MYHs.
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