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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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Characterization of pancreatic tissue expressing tumorigenic and non-tumorigenic KRASmutant alleles(A) Pancreatic tissue from uninjected control ptf1a:Gal4-VP16 fish had histologically normal pancreas and no evidence of tumor formation in any organ. Control pancreatic tissue also displayed no labeling for eGFP and minimal labeling for p-ERK and PCNA. (B-E) Representative pancreatic tissue from fish injected with tumorigenic mutations G12C, G12D, G12R and G12V. Identical results were also observed for fish injected with G13D, Q61L, Q61R, and A146T (data not shown). Resulting tumors were uniformly positive for eGFP and showed strong labeling for p-ERK and PCNA. (F-I) Representative pancreatic tissue from fish injected with non-tumorigenic mutations G12A, G12F, G12S, and G13C. In spite of widespread expression of eGFP-KRASmutant transgenes, normal histology and minimal labeling for p-ERK and PCNA are observed. Regions outlined by dotted lines indicated areas depicted at higher magnification in adjacent images. Primary antibodies used for immunohistochemistry were rabbit anti-eGFP (Invitrogen, A11122, 1:400), rabbit anti-phospho-ERK (Cell Signaling Technology, 4370S, 1:400), and mouse anti-PCNA (DAKO, M0879, 1:400).
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Figure 4: Characterization of pancreatic tissue expressing tumorigenic and non-tumorigenic KRASmutant alleles(A) Pancreatic tissue from uninjected control ptf1a:Gal4-VP16 fish had histologically normal pancreas and no evidence of tumor formation in any organ. Control pancreatic tissue also displayed no labeling for eGFP and minimal labeling for p-ERK and PCNA. (B-E) Representative pancreatic tissue from fish injected with tumorigenic mutations G12C, G12D, G12R and G12V. Identical results were also observed for fish injected with G13D, Q61L, Q61R, and A146T (data not shown). Resulting tumors were uniformly positive for eGFP and showed strong labeling for p-ERK and PCNA. (F-I) Representative pancreatic tissue from fish injected with non-tumorigenic mutations G12A, G12F, G12S, and G13C. In spite of widespread expression of eGFP-KRASmutant transgenes, normal histology and minimal labeling for p-ERK and PCNA are observed. Regions outlined by dotted lines indicated areas depicted at higher magnification in adjacent images. Primary antibodies used for immunohistochemistry were rabbit anti-eGFP (Invitrogen, A11122, 1:400), rabbit anti-phospho-ERK (Cell Signaling Technology, 4370S, 1:400), and mouse anti-PCNA (DAKO, M0879, 1:400).

Mentions: We next sacrificed adult fish with or without transcutaneous eGFP fluorescence in the cerebellum, and examined pancreatic transgene expression as assessed by eGFP fluorescence within dissected abdominal viscera. As in the case of cerebellum, all twelve versions of activated KRAS were associated with significant frequencies of pancreatic eGFP fluorescence, ranging from 8%-66.7%. Representative transcutaneous and pancreatic eGFP fluorescence for G12D and G12V are shown in Fig. 2B,C and Fig 2J,K, and additional examples of are shown in Supplemental Fig. S1. Rates of pancreatic eGFP fluorescence are depicted in Fig. 3 (dark green bars), and further immunohistochemical confirmation of eGFP-KRASmutant transgene expression is provided in Figure 4.


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)

Characterization of pancreatic tissue expressing tumorigenic and non-tumorigenic KRASmutant alleles(A) Pancreatic tissue from uninjected control ptf1a:Gal4-VP16 fish had histologically normal pancreas and no evidence of tumor formation in any organ. Control pancreatic tissue also displayed no labeling for eGFP and minimal labeling for p-ERK and PCNA. (B-E) Representative pancreatic tissue from fish injected with tumorigenic mutations G12C, G12D, G12R and G12V. Identical results were also observed for fish injected with G13D, Q61L, Q61R, and A146T (data not shown). Resulting tumors were uniformly positive for eGFP and showed strong labeling for p-ERK and PCNA. (F-I) Representative pancreatic tissue from fish injected with non-tumorigenic mutations G12A, G12F, G12S, and G13C. In spite of widespread expression of eGFP-KRASmutant transgenes, normal histology and minimal labeling for p-ERK and PCNA are observed. Regions outlined by dotted lines indicated areas depicted at higher magnification in adjacent images. Primary antibodies used for immunohistochemistry were rabbit anti-eGFP (Invitrogen, A11122, 1:400), rabbit anti-phospho-ERK (Cell Signaling Technology, 4370S, 1:400), and mouse anti-PCNA (DAKO, M0879, 1:400).
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Related In: Results  -  Collection

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

Figure 4: Characterization of pancreatic tissue expressing tumorigenic and non-tumorigenic KRASmutant alleles(A) Pancreatic tissue from uninjected control ptf1a:Gal4-VP16 fish had histologically normal pancreas and no evidence of tumor formation in any organ. Control pancreatic tissue also displayed no labeling for eGFP and minimal labeling for p-ERK and PCNA. (B-E) Representative pancreatic tissue from fish injected with tumorigenic mutations G12C, G12D, G12R and G12V. Identical results were also observed for fish injected with G13D, Q61L, Q61R, and A146T (data not shown). Resulting tumors were uniformly positive for eGFP and showed strong labeling for p-ERK and PCNA. (F-I) Representative pancreatic tissue from fish injected with non-tumorigenic mutations G12A, G12F, G12S, and G13C. In spite of widespread expression of eGFP-KRASmutant transgenes, normal histology and minimal labeling for p-ERK and PCNA are observed. Regions outlined by dotted lines indicated areas depicted at higher magnification in adjacent images. Primary antibodies used for immunohistochemistry were rabbit anti-eGFP (Invitrogen, A11122, 1:400), rabbit anti-phospho-ERK (Cell Signaling Technology, 4370S, 1:400), and mouse anti-PCNA (DAKO, M0879, 1:400).
Mentions: We next sacrificed adult fish with or without transcutaneous eGFP fluorescence in the cerebellum, and examined pancreatic transgene expression as assessed by eGFP fluorescence within dissected abdominal viscera. As in the case of cerebellum, all twelve versions of activated KRAS were associated with significant frequencies of pancreatic eGFP fluorescence, ranging from 8%-66.7%. Representative transcutaneous and pancreatic eGFP fluorescence for G12D and G12V are shown in Fig. 2B,C and Fig 2J,K, and additional examples of are shown in Supplemental Fig. S1. Rates of pancreatic eGFP fluorescence are depicted in Fig. 3 (dark green bars), and further immunohistochemical confirmation of eGFP-KRASmutant transgene expression is provided in Figure 4.

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.

Show MeSH
Related in: MedlinePlus