TY - JOUR
T1 - The canonical Wnt pathway regulates the metastasis-promoting mucin MUC4 in pancreatic ductal adenocarcinoma
AU - Pai, Priya
AU - Rachagani, Satyanarayana
AU - Lakshmanan, Imayavaramban
AU - Macha, Muzafar A
AU - Sheinin, Yuri
AU - Smith, Lynette M.
AU - Ponnusamy, Moorthy P.
AU - Batra, Surinder K.
N1 - Funding Information:
This work was supported, in part, by the grants from National Institutes of Health ( UO1 CA111294 , P50 CA127297 , U54 CA163120 and RO1 CA183459 ). The authors would like to thank Ms. Kavita Mallya, Dr. Parthasarathy Seshacharyulu, Dr. Sushil Kumar, Dr. Arokiapriyanka Vaz and Suprit Gupta for technical support; Ms. Michelle Varney for the lentiviral transfection protocol; Drs. Robert Bennett (UNMC) and Yuzuru Shiio (UTHSC) for plasmids donated; Dr. Jing (Jenny) Wang (UNMC) for the L Wnt3A cells; Drs. Angie Rizzino and Shilpa Buch for letting us use their facilities; UNMC DNA Sequencing Core Facility, and Janice A. Taylor and James R. Talaska of the UNMC confocal laser scanning facility for assistance with taking confocal images. Finally, we kindly thank Melody A. Montgomery, BS, MFA, for the professional editing of this manuscript.
Publisher Copyright:
© 2015 .
PY - 2016/2/1
Y1 - 2016/2/1
N2 - Aberrant Wnt signaling frequently occurs in pancreatic cancer (PC) and contributes to disease progression/metastases. Likewise, the transmembrane-mucin MUC4 is expressed de novo in early pancreatic intraepithelial neoplasia (PanINs) and incrementally increases with PC progression, contributing to metastasis. To determine the mechanism of MUC4 upregulation in PC, we examined factors deregulated in early PC progression, such as Wnt/β-catenin signaling. MUC4 promoter analysis revealed the presence of three putative TCF/LEF-binding sites, leading us to hypothesize that MUC4 can be regulated by β-catenin. Immunohistochemical (IHC) analysis of rapid autopsy PC tissues showed a correlation between MUC4 and cytosolic/nuclear β-catenin expression. Knock down (KD) of β-catenin in CD18/HPAF and T3M4 cell lines resulted in decreased MUC4 transcript and protein. Three MUC4 promoter luciferase constructs, p3778, p3000, and p2700, were generated. The construct p3778, encompassing the entire MUC4 promoter, elicited increased luciferase activity in the presence of stabilized β-catenin. Mutation of the TCF/LEF site closest to the transcription start site (i.e., -2629/-2612) and furthest from the start site (i.e., -3425/-3408) reduced MUC4 promoter luciferase activity. Transfection with dominant negative TCF4 decreased MUC4 transcript and protein levels. Chromatin immunoprecipitation confirmed enrichment of β-catenin on -2629/-2612 and -3425/-3408 of the MUC4 promoter in CD18/HPAF. Functionally, CD18/HPAF and T3M4 β-catenin KD cells showed decreased migration and decreased Vimentin, N-cadherin, and pERK1/2 expression. Tumorigenicity studies in athymic nude mice showed CD18/HPAF β-catenin KD cells significantly reduced primary tumor sizes and metastases compared to scrambled control cells. We show for the first time that β-catenin directly governs MUC4 in PC.
AB - Aberrant Wnt signaling frequently occurs in pancreatic cancer (PC) and contributes to disease progression/metastases. Likewise, the transmembrane-mucin MUC4 is expressed de novo in early pancreatic intraepithelial neoplasia (PanINs) and incrementally increases with PC progression, contributing to metastasis. To determine the mechanism of MUC4 upregulation in PC, we examined factors deregulated in early PC progression, such as Wnt/β-catenin signaling. MUC4 promoter analysis revealed the presence of three putative TCF/LEF-binding sites, leading us to hypothesize that MUC4 can be regulated by β-catenin. Immunohistochemical (IHC) analysis of rapid autopsy PC tissues showed a correlation between MUC4 and cytosolic/nuclear β-catenin expression. Knock down (KD) of β-catenin in CD18/HPAF and T3M4 cell lines resulted in decreased MUC4 transcript and protein. Three MUC4 promoter luciferase constructs, p3778, p3000, and p2700, were generated. The construct p3778, encompassing the entire MUC4 promoter, elicited increased luciferase activity in the presence of stabilized β-catenin. Mutation of the TCF/LEF site closest to the transcription start site (i.e., -2629/-2612) and furthest from the start site (i.e., -3425/-3408) reduced MUC4 promoter luciferase activity. Transfection with dominant negative TCF4 decreased MUC4 transcript and protein levels. Chromatin immunoprecipitation confirmed enrichment of β-catenin on -2629/-2612 and -3425/-3408 of the MUC4 promoter in CD18/HPAF. Functionally, CD18/HPAF and T3M4 β-catenin KD cells showed decreased migration and decreased Vimentin, N-cadherin, and pERK1/2 expression. Tumorigenicity studies in athymic nude mice showed CD18/HPAF β-catenin KD cells significantly reduced primary tumor sizes and metastases compared to scrambled control cells. We show for the first time that β-catenin directly governs MUC4 in PC.
KW - MUC4
KW - Pancreatic cancer
KW - Wnt
KW - β-Catenin
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U2 - 10.1016/j.molonc.2015.10.005
DO - 10.1016/j.molonc.2015.10.005
M3 - Article
C2 - 26526617
AN - SCOPUS:84958924067
SN - 1574-7891
VL - 10
SP - 224
EP - 239
JO - Molecular Oncology
JF - Molecular Oncology
IS - 2
ER -