Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice

Seema Chugh, Srikanth Barkeer, Satyanarayana Rachagani, Rama Krishna Nimmakayala, Naveenkumar Perumal, Ramesh Pothuraju, Pranita Atri, Sidharth Mahapatra, Ishwor Thapa, Geoffrey A Talmon, Lynette M Smith, Xinheng Yu, Sriram Neelamegham, Jianxin Fu, Lijun Xia, Moorthy Palanimuthu Ponnusamy, Surinder Kumar Batra

Research output: Contribution to journalArticle

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Abstract

Background & Aims: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. Methods: We crossed C1galt1 floxed mice (C1galt1 loxP/loxP ) with Kras G12D/+ ; Trp53 R172H/+ ; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. Results: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. Conclusions: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

Original languageEnglish (US)
Pages (from-to)1608-1624
Number of pages17
JournalGastroenterology
Volume155
Issue number5
DOIs
StatePublished - Nov 2018

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Adenocarcinoma
Neoplasm Metastasis
Genes
Polysaccharides
Lectins
Glycosylation
Clustered Regularly Interspaced Short Palindromic Repeats
Immunohistochemistry
RNA Sequence Analysis
Galactosyltransferases
Acetylgalactosamine
Neoplasms
Alcian Blue
Fluorescent Antibody Technique
Pancreas
Mass Spectrometry
Glycoproteins
Carcinogenesis
Staining and Labeling
Gene Expression

Keywords

  • Mouse Model
  • Pancreas
  • Pancreatic Intraepithelial Neoplasias
  • Post-Translational Modification

ASJC Scopus subject areas

  • Hepatology
  • Gastroenterology

Cite this

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice. / Chugh, Seema; Barkeer, Srikanth; Rachagani, Satyanarayana; Nimmakayala, Rama Krishna; Perumal, Naveenkumar; Pothuraju, Ramesh; Atri, Pranita; Mahapatra, Sidharth; Thapa, Ishwor; Talmon, Geoffrey A; Smith, Lynette M; Yu, Xinheng; Neelamegham, Sriram; Fu, Jianxin; Xia, Lijun; Palanimuthu Ponnusamy, Moorthy; Batra, Surinder Kumar.

In: Gastroenterology, Vol. 155, No. 5, 11.2018, p. 1608-1624.

Research output: Contribution to journalArticle

Chugh, S, Barkeer, S, Rachagani, S, Nimmakayala, RK, Perumal, N, Pothuraju, R, Atri, P, Mahapatra, S, Thapa, I, Talmon, GA, Smith, LM, Yu, X, Neelamegham, S, Fu, J, Xia, L, Palanimuthu Ponnusamy, M & Batra, SK 2018, 'Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice', Gastroenterology, vol. 155, no. 5, pp. 1608-1624. https://doi.org/10.1053/j.gastro.2018.08.007
Chugh, Seema ; Barkeer, Srikanth ; Rachagani, Satyanarayana ; Nimmakayala, Rama Krishna ; Perumal, Naveenkumar ; Pothuraju, Ramesh ; Atri, Pranita ; Mahapatra, Sidharth ; Thapa, Ishwor ; Talmon, Geoffrey A ; Smith, Lynette M ; Yu, Xinheng ; Neelamegham, Sriram ; Fu, Jianxin ; Xia, Lijun ; Palanimuthu Ponnusamy, Moorthy ; Batra, Surinder Kumar. / Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice. In: Gastroenterology. 2018 ; Vol. 155, No. 5. pp. 1608-1624.
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TY - JOUR

T1 - Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice

AU - Chugh, Seema

AU - Barkeer, Srikanth

AU - Rachagani, Satyanarayana

AU - Nimmakayala, Rama Krishna

AU - Perumal, Naveenkumar

AU - Pothuraju, Ramesh

AU - Atri, Pranita

AU - Mahapatra, Sidharth

AU - Thapa, Ishwor

AU - Talmon, Geoffrey A

AU - Smith, Lynette M

AU - Yu, Xinheng

AU - Neelamegham, Sriram

AU - Fu, Jianxin

AU - Xia, Lijun

AU - Palanimuthu Ponnusamy, Moorthy

AU - Batra, Surinder Kumar

PY - 2018/11

Y1 - 2018/11

N2 - Background & Aims: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. Methods: We crossed C1galt1 floxed mice (C1galt1 loxP/loxP ) with Kras G12D/+ ; Trp53 R172H/+ ; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. Results: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. Conclusions: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

AB - Background & Aims: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. Methods: We crossed C1galt1 floxed mice (C1galt1 loxP/loxP ) with Kras G12D/+ ; Trp53 R172H/+ ; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. Results: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. Conclusions: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

KW - Mouse Model

KW - Pancreas

KW - Pancreatic Intraepithelial Neoplasias

KW - Post-Translational Modification

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U2 - 10.1053/j.gastro.2018.08.007

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