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Differential in vivo tumorigenicity of diverse KRAS mutations in vertebrate pancreas: A comprehensive survey.

Park JT, Johnson N, Liu S, Levesque M, Wang YJ, Ho H, Huso D, Maitra A, Parsons MJ, Prescott JD, Leach SD - Oncogene (2014)

Bottom Line: These mutations occur primarily at codon 12 and less frequently at codons 13 and 61.All eight tumorigenic KRAS mutations were associated with downstream MAPK/ERK pathway activation in preneoplastic pancreatic epithelium, whereas nontumorigenic mutations were not.These results suggest that the spectrum of KRAS mutations observed in human pancreatic cancer reflects selection based on variable tumorigenic capacities, including the ability to activate MAPK/ERK signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Johns Hopkins Medical Institutions, Baltimore, MD, USA.

ABSTRACT
Somatic activation of the KRAS proto-oncogene is evident in almost all pancreatic cancers, and appears to represent an initiating event. These mutations occur primarily at codon 12 and less frequently at codons 13 and 61. Although some studies have suggested that different KRAS mutations may have variable oncogenic properties, to date there has been no comprehensive functional comparison of multiple KRAS mutations in an in vivo vertebrate tumorigenesis system. We generated a Gal4/UAS-based zebrafish model of pancreatic tumorigenesis in which the pancreatic expression of UAS-regulated oncogenes is driven by a ptf1a:Gal4-VP16 driver line. This system allowed us to rapidly compare the ability of 12 different KRAS mutations (G12A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, G13D, Q61L, Q61R and A146T) to drive pancreatic tumorigenesis in vivo. Among fish injected with one of five KRAS mutations reported in other tumor types but not in human pancreatic cancer, 2/79 (2.5%) developed pancreatic tumors, with both tumors arising in fish injected with A146T. In contrast, among fish injected with one of seven KRAS mutations known to occur in human pancreatic cancer, 22/106 (20.8%) developed pancreatic cancer. All eight tumorigenic KRAS mutations were associated with downstream MAPK/ERK pathway activation in preneoplastic pancreatic epithelium, whereas nontumorigenic mutations were not. These results suggest that the spectrum of KRAS mutations observed in human pancreatic cancer reflects selection based on variable tumorigenic capacities, including the ability to activate MAPK/ERK signaling.

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Targeted expression of eGFP-KRASmutant transgene in zebrafish pancreas(A) Schematic depiction of experimental design employing the Gal4-VP16/UAS system used to drive eGFP-KRASmutant transgene expression within the ptf1a expression domain. (B-C) Lateral view of larval zebrafish (5dpf) under transmitted and fluorescent illumination, showing expression pattern of eGFP-KRASmutant transgene in the hindbrain. Due to yolk autofluorescence (asterisk *), pancreatic expression of the eGFP-KRASmutant transgene is difficult to detect in intact embyos. (D-G) Confocal image of microdissected pancreas from 5 dpf larval fish, revealing the membrane localization of eGFP-KRASmutant protein. Blue pseudocolor indicates DAPI labeling.
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Figure 1: Targeted expression of eGFP-KRASmutant transgene in zebrafish pancreas(A) Schematic depiction of experimental design employing the Gal4-VP16/UAS system used to drive eGFP-KRASmutant transgene expression within the ptf1a expression domain. (B-C) Lateral view of larval zebrafish (5dpf) under transmitted and fluorescent illumination, showing expression pattern of eGFP-KRASmutant transgene in the hindbrain. Due to yolk autofluorescence (asterisk *), pancreatic expression of the eGFP-KRASmutant transgene is difficult to detect in intact embyos. (D-G) Confocal image of microdissected pancreas from 5 dpf larval fish, revealing the membrane localization of eGFP-KRASmutant protein. Blue pseudocolor indicates DAPI labeling.

Mentions: To functionally compare the ability of different human KRAS mutations to initiate pancreatic tumorigenesis, twelve different mutations (G12A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, G13D, Q61L, Q61R, and A146T) were selected for analysis. KRAS mutant alleles were generated by modifying a wild-type human KRAS cDNA using site-directed mutatgenesis followed by full length sequencing to confirm successful mutation. Each mutant variant was expressed as an eGFP-KRASmutant fusion under the transcriptional control of a concatamerized 14×UAS element.9 Mosaic pancreatic expression was achieved by injection of UAS:eGFP-KRASmutant constructs into hemizygous ptf1a:Gal4-VP16 transgenic embryos produced from a cross between the Tg(ptf1a:Gal4-VP16)JH16 BAC transgenic line10 and wildtype AB fish (Fig. 1A). Reflecting known patterns of ptf1a gene expression, eGFP expression was first observed in the developing hindbrain and cerebellum (Fig. 1B,C). Due to yolk autofluorescence, pancreas-specific expression of eGFP-KRASmutant transgenes proved to be difficult to detect in whole mount embryos (Fig. 1B and C, asterisk). However, confocal imaging of the micro-dissected pancreas from 5 dpf larvae revealed effective expression and membrane localization of the eGFP-KRASmutant protein (Fig. 1D-G). On day five, embryos showing eGFP fluorescence within the ptf1a expression domain were selected and raised to adulthood.


Differential in vivo tumorigenicity of diverse KRAS mutations in vertebrate pancreas: A comprehensive survey.

Park JT, Johnson N, Liu S, Levesque M, Wang YJ, Ho H, Huso D, Maitra A, Parsons MJ, Prescott JD, Leach SD - Oncogene (2014)

Targeted expression of eGFP-KRASmutant transgene in zebrafish pancreas(A) Schematic depiction of experimental design employing the Gal4-VP16/UAS system used to drive eGFP-KRASmutant transgene expression within the ptf1a expression domain. (B-C) Lateral view of larval zebrafish (5dpf) under transmitted and fluorescent illumination, showing expression pattern of eGFP-KRASmutant transgene in the hindbrain. Due to yolk autofluorescence (asterisk *), pancreatic expression of the eGFP-KRASmutant transgene is difficult to detect in intact embyos. (D-G) Confocal image of microdissected pancreas from 5 dpf larval fish, revealing the membrane localization of eGFP-KRASmutant protein. Blue pseudocolor indicates DAPI labeling.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836617&req=5

Figure 1: Targeted expression of eGFP-KRASmutant transgene in zebrafish pancreas(A) Schematic depiction of experimental design employing the Gal4-VP16/UAS system used to drive eGFP-KRASmutant transgene expression within the ptf1a expression domain. (B-C) Lateral view of larval zebrafish (5dpf) under transmitted and fluorescent illumination, showing expression pattern of eGFP-KRASmutant transgene in the hindbrain. Due to yolk autofluorescence (asterisk *), pancreatic expression of the eGFP-KRASmutant transgene is difficult to detect in intact embyos. (D-G) Confocal image of microdissected pancreas from 5 dpf larval fish, revealing the membrane localization of eGFP-KRASmutant protein. Blue pseudocolor indicates DAPI labeling.
Mentions: To functionally compare the ability of different human KRAS mutations to initiate pancreatic tumorigenesis, twelve different mutations (G12A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, G13D, Q61L, Q61R, and A146T) were selected for analysis. KRAS mutant alleles were generated by modifying a wild-type human KRAS cDNA using site-directed mutatgenesis followed by full length sequencing to confirm successful mutation. Each mutant variant was expressed as an eGFP-KRASmutant fusion under the transcriptional control of a concatamerized 14×UAS element.9 Mosaic pancreatic expression was achieved by injection of UAS:eGFP-KRASmutant constructs into hemizygous ptf1a:Gal4-VP16 transgenic embryos produced from a cross between the Tg(ptf1a:Gal4-VP16)JH16 BAC transgenic line10 and wildtype AB fish (Fig. 1A). Reflecting known patterns of ptf1a gene expression, eGFP expression was first observed in the developing hindbrain and cerebellum (Fig. 1B,C). Due to yolk autofluorescence, pancreas-specific expression of eGFP-KRASmutant transgenes proved to be difficult to detect in whole mount embryos (Fig. 1B and C, asterisk). However, confocal imaging of the micro-dissected pancreas from 5 dpf larvae revealed effective expression and membrane localization of the eGFP-KRASmutant protein (Fig. 1D-G). On day five, embryos showing eGFP fluorescence within the ptf1a expression domain were selected and raised to adulthood.

Bottom Line: These mutations occur primarily at codon 12 and less frequently at codons 13 and 61.All eight tumorigenic KRAS mutations were associated with downstream MAPK/ERK pathway activation in preneoplastic pancreatic epithelium, whereas nontumorigenic mutations were not.These results suggest that the spectrum of KRAS mutations observed in human pancreatic cancer reflects selection based on variable tumorigenic capacities, including the ability to activate MAPK/ERK signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Johns Hopkins Medical Institutions, Baltimore, MD, USA.

ABSTRACT
Somatic activation of the KRAS proto-oncogene is evident in almost all pancreatic cancers, and appears to represent an initiating event. These mutations occur primarily at codon 12 and less frequently at codons 13 and 61. Although some studies have suggested that different KRAS mutations may have variable oncogenic properties, to date there has been no comprehensive functional comparison of multiple KRAS mutations in an in vivo vertebrate tumorigenesis system. We generated a Gal4/UAS-based zebrafish model of pancreatic tumorigenesis in which the pancreatic expression of UAS-regulated oncogenes is driven by a ptf1a:Gal4-VP16 driver line. This system allowed us to rapidly compare the ability of 12 different KRAS mutations (G12A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, G13D, Q61L, Q61R and A146T) to drive pancreatic tumorigenesis in vivo. Among fish injected with one of five KRAS mutations reported in other tumor types but not in human pancreatic cancer, 2/79 (2.5%) developed pancreatic tumors, with both tumors arising in fish injected with A146T. In contrast, among fish injected with one of seven KRAS mutations known to occur in human pancreatic cancer, 22/106 (20.8%) developed pancreatic cancer. All eight tumorigenic KRAS mutations were associated with downstream MAPK/ERK pathway activation in preneoplastic pancreatic epithelium, whereas nontumorigenic mutations were not. These results suggest that the spectrum of KRAS mutations observed in human pancreatic cancer reflects selection based on variable tumorigenic capacities, including the ability to activate MAPK/ERK signaling.

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Related in: MedlinePlus