Identification of functionally distinct TRAF proinflammatory and phosphatidylinositol 3-kinase/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (PI3K/MEK) transforming activities emanating from RET/PTC fusion oncoprotein

Abstract

Thyroid carcinomas that harbor RET/PTC oncogenes are well differentiated, relatively benign neoplasms compared with those expressing oncogenic RAS or BRAF mutations despite signaling through shared transforming pathways. A distinction, however, is that RET/PTCs induce immunostimulatory programs, suggesting that, in the case of this tumor type, the additional pro-inflammatory pathway reduces aggressiveness. Here, we demonstrate that pro-inflammatory programs are selectively activated by TRAF2 and TRAF6 association with RET/PTC oncoproteins. Eliminating this mechanism reduces pro-inflammatory cytokine production without decreasing transformation efficiency. Conversely, ablating MEK/ERK or PI3K/AKT signaling eliminates transformation but not pro-inflammatory cytokine secretion. Functional uncoupling of the two pathways demonstrates that intrinsic pro-inflammatory pathways are not required for cellular transformation and suggests a need for further investigation into the role inflammation plays in thyroid tumor progression.

FIGURE 1.

Inhibitors of PI3K or MEK do not inhibit RP3-mediated proinflammatory cytokine transcription. A, PCCL3 cells were transiently transfected with RP3^WT or RP3^K284M in the presence of 0.1% DMSO alone, 10 μm LY294002, or 10 μm of U0126 for 48 h, and cells were lysed for total RNA followed by reverse transcription and PCR for rat-specific gene products. B, PCCL3 cells were treated as in A and harvested at 48 h for total protein and Western blot analysis. C, TPC-1 cells were treated for 48 h with 0.1% DMSO alone, 10 μm LY294002, or 10 μm U0126 and subsequently harvested for total RNA followed by reverse transcription and PCR for human-specific gene products. D, TPC-1 cells were treated as in C and harvested at 48 h for total protein and Western blot analysis. A–D, data representative of two independent experiments. RevT, reverse transcriptase; LY, LY294002; U0, U0126.

FIGURE 2.

A and B, RP3^Y478F exhibits reduced MCP-1 expression and maintains AKT and ERK activation. A, PCCL3 cells were transiently transfected with Rc/CMV vector alone, RP3^WT, or RP3^Tyr→Phe mutants for 48 h, and cells were lysed; total RNA was extracted and RNA reverse-transcribed, and cDNA was amplified using rat GAPDH- and MCP1-specific primers. Error bars indicate S.E. from at least three independent experiments. B, PCCL3 cells were transiently transfected with MSCV.IRES.GFP vector alone, RP3^WT, RP3^K284M, or RP3^Y588F/Y622F mutants for 48 h, and cells were lysed; total RNA was extracted and RNA reverse-transcribed, and cDNA was amplified using rat GAPDH- and MCP1-specific primers. C, representative Western blot of two individual experiments of PCCL3 cells that were treated as in A and harvested at 48 h for total protein.

FIGURE 3.

RP3 associates with TRAF2 and TRAF6 in a dimer-dependent and kinase-independent manner. A, schematic representation of RP3^WT, RP3^K284M, and truncation mutants. B–F, 293T cells were transiently transfected with GFP, RP3^WT, or RP3^K284M or truncation (Δ) mutants for 48 h, lysed, and subsequently harvested for immunoprecipitation using an anti-RET.51-specific antibody. Immunoprecipitated protein was subjected to Western blot analysis using antibodies specific for TRAF2, TRAF6, RET, or actin. G, TPC-1 cell were lysed and subsequently harvested for immunoprecipitation using an anti-RET.51-specific antibody. Immunoprecipitated protein was subjected to Western blot analysis using antibodies specific for TRAF2, TRAF6, RET, or actin. Data are representative of at three independent experiments. IP, immunoprecipitation; UB, unbound fraction; hIgG, goat IgG heavy chain.

FIGURE 4.

Knockdown of TRAF2 and TRAF6 abrogates cytokine expression. A and B, TPC-1 cells were transfected with nontargeting control, TRAF2, or TRAF6 siRNA and harvested after 48 h for total RNA followed by reverse transcription and PCR using human GAPDH-, MCP1-, IL6-, IL8-, TNF-, GM-CSF-, TRAF2-, TRAF6-, and RET/PTC1-specific primers. A, representative experiment of cytokine transcript induction of at least three individual experiments. B, graphs are represented as densitometric analysis of band intensity relative to GAPDH and the fold induction of that ratio over scrambled siRNA control. Error bars indicate S.D. of samples run in triplicate, in three independent experiments. C, MCP1 secretion was measured on day 4 by ELISA using culture supernatants derived from TPC-1 cells transfected with scrambled control, TRAF2, or TRAF6 siRNAs treated on day 0 and re-treated on day 2. D, TPC-1 cells were transduced with MSCV.IRES.RFP constructs expressing T2- and T6-binding motifs and their peptide controls. Following selection for RFP, cells were harvested for total RNA followed by reverse transcription and PCR using human GAPDH- and MCP1-specific primers. E, PCCL3 cells were stably transduced with RP3^WT or RP3^Tyr→Phe mutants, and after a month of passage in culture, cells were plated overnight and lysed for total RNA followed by reverse transcription and PCR with rat GAPDH- and MCP-1-specific primers. Data are represented as densitometric measurements of band intensity relative to GAPDH and the fold induction of that ratio over RP3. C–E, error bars indicate S.D. of samples run in duplicate in two independent experiments. Cntl, nontargeting siRNA control; T2, TRAF2; T6, TRAF6.

FIGURE 5.

Disruption of TRAF2 and -6 pathways does not inhibit AKT and ERK pathways. A, TPC-1 cells were transfected with scrambled control, TRAF2, or TRAF6 siRNA and re-treated with siRNA and on day 2 and 4 were harvested for Western blot analysis. B, TPC-1 cells were transduced with MSCV.IRES.RFP constructs expressing T2- and T6-binding motifs and their peptide controls. Following selection for RFP, cells were harvested for Western blot analysis. C, cell proliferation assay of transduced TPC-1 cells from B that were plated at 1000 cells/well in 96-well culture dishes and counted daily. D, ERK-dependent hormone-independent cell proliferation assay of transduced PCCL3 cells expressing wild type RP3 and mutants. Cells were plated at 1000 cells/well in 96-well culture dishes in the absence of six hormones (TSH, insulin, Gly-His-Lys, somatostatin, apotransferrin, and hydrocortisone) and counted daily. Data are representative of three independent experiments. Cntl, nontargeting siRNA control; T2, TRAF2; T6, TRAF6.

FIGURE 6.

TRAF-deficient RP3 mutants maintain transforming activity. A, representative photographs of anchorage-independent colony growth of NIH-3T3 cells stably transduced with RP3^WT and mutants used for the analysis in B. B, number of colonies counted from a defined grid that were produced from NIH-3T3 transductants as in A that were greater than 100 μm in length and averaged over triplicate wells. The table below the graph represents the average colony length and standard deviation of the triplicate wells. Data are representative of two independent experiments.

FIGURE 7.

Model of RET/PTC-mediated TRAF activation. RET/PTC dimers exhibit a greater affinity for TRAF2 and TRAF6 than RET/PTC monomers. The TRAF interaction with RET/PTC does not require an active conformation of the kinase domain. However, TRAF activation does require an active kinase domain conformation, which most likely induces TRAF oligomerization to promote NIK and NF-κB-mediated proinflammatory signaling. The RET/PTC-TRAF-NIK-NF-κB pathway is functionally distinct from Tyr-588-mediated RAS and PI3K signaling cascades that promote transformation.