Transcriptome analysis in mouse tumors induced by Ret-MEN2/FMTC mutations reveals subtype-specific role in survival and interference with immune surveillance

Abstract

Activating mutations in the Ret proto-oncogene are responsible for occurrence of multiple endocrine neoplasia (MEN) type 2A and 2B, and familial medullary thyroid carcinoma (FMTC). A striking genotype-phenotype correlation between the mutated RET codon and clinical manifestation implies that tumorigenesis is conditioned by the type of mutation. We investigated gene expression profiles between and within distinct MEN2 subtypes through whole-genome microarray analysis in tumors induced by NIH-3T3 cells transformed with defined RET-MEN2A (C609Y, C634R), MEN2B, (A883F, M918T), and FMTC (Y791F) mutations. Expression profiling identified a statistically significant modification of 1494 genes, 628 down-and 866 upregulated in MEN2B compared with MEN2A/FMTC tumors. By contrast, no obvious alterations were observed among individual MEN2B and MEN2A type mutations, or between MEN2A and FMTC. Functional clustering of differential genes revealed RET-MEN2B specific upregulation of genes associated with novel growth and survival pathways. Intriguingly, RET-MEN2A/FMTC-specific tumors were characterized by a considerable number of genes involved in the host antitumor immune response via stimulation of natural killer/T-cell proliferation, migration, and cytotoxicity, which were completely absent in RET-MEN2B related cancers. QPCR on tumors versus cultured NIH-RET cell lines demonstrated that they are largely attributed to the host innate immune system, whereas expression of CX3CL1 involved in leukocyte recruitment is exclusively RET-MEN2A/FMTC tumor cell dependent. In correlation, massive inflammatory infiltrates were apparent only in tumors carrying MEN type 2A/FMTC mutations, suggesting that RET-MEN2B receptors specifically counteract immune infiltration by preventing chemokine expression, which may contribute to the different clinical outcome of both subtypes.

STAT3 negatively regulates thyroid tumorigenesis

“…Studies have also shown that oncogenic RET can increase proinflammatory mediators, such as IL-6, in thyroid cells and tumors (46)(47)(48). In addition, BRAFV600E has been shown to induce the secretion of IL-6 in melanoma cell lines (24).…”

RET-mediated modulation of tumor microenvironment and immune response in multiple endocrine neoplasia type 2 (MEN2)

“…An IHC analysis of these tumors confirmed that granzyme and perforin1 antibodies could stain the immune infiltrate in RET MEN2A-, but not RET MEN2B-induced tumors. Consistently, the authors observed that RET MEN2A/FMTC, but not RET MEN2B tumors expressed CX3CL1/fractalkine, a chemokine involved in the recruitment of cytotoxic immune cells, into both experimental tumor models and in human cancer tissues (Engelmann et al 2009). These data suggest two possible interpretations: 1.…”

“…RET oncogenic proteins can activate, albeit constitutively and more efficiently, the same signaling pathways induced by physiologically ligand-stimulated RET. However, some differences in downstream signaling between wild-type RET and different MEN2 mutants may account for differential gene expression, that can be translated in differences in tumor microenvironment, and ultimately in disease severity in MTC (Engelmann et al 2009). …”

“…Many investigators have used mice models to study RET-mediated immunomodulatory activity. Interestingly, in a study comparing gene expression profiles associated with RET MEN2A, RET MEN2B and RET FMTC mutations, Engelman and coworkers observed that a signature of genes related to the host immuneinflammatory response, related principally to NK cells and cytotoxic T lymphocytes (CTL), were enriched in RET MEN2A and FMTC tumors with respect to RET MEN2B (Engelmann et al 2009). The authors also hypothesized that RET MEN2A/FMTC-induced tumors were more susceptible to cytotoxic cells than RET MEN2B tumors.…”

Evaluation of systemic targeting of RET oncogene-based MTC with tumor-selective peptide-tagged Ad vectors in clinical mouse models

“…5 This causes ligand-independent homodimerization and constitutive autophosphorylation, and activation of the RET kinase, leading to the stimulation of a complex network of signal transduction pathways that contribute to dysregulated growth and survival. 7,8 Activated oncogenes such as RET are highly attractive targets for the development of anticancer agents. 9 Principles of RET inhibition in MTC cells include molecular strategies to interfere with its kinase activity by small molecules [10][11][12] and receptor dimerization using aptamers or soluble RET ectodomain.…”