Loss of Tumour Suppressor TMEM127 Drives RET-mediated Transformation Through Disrupted Membrane Dynamics

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    This study highlights new insights into the mechanism of pheochromocytoma pathogenesis that remains poorly understood. In the context of hereditary syndromes, such as multiple endocrine neoplasia 2 (MEN-2), where RET mutation is the major driver of thyroid, parathyroid, and adrenal pathologies, including pheochromocytoma, this mechanistic dissection of RET and TMEM127 is fundamentally sound. While the significance was deemed important, the strength of the evidence was found to be solid, although additional validation work would strengthen the findings.

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Abstract

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTK) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumour pheochromocytoma (PCC) can be caused by activating mutations of the RET receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumour suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization, including membrane protein diffusability and protein complex assembly, and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

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  1. eLife assessment

    This study highlights new insights into the mechanism of pheochromocytoma pathogenesis that remains poorly understood. In the context of hereditary syndromes, such as multiple endocrine neoplasia 2 (MEN-2), where RET mutation is the major driver of thyroid, parathyroid, and adrenal pathologies, including pheochromocytoma, this mechanistic dissection of RET and TMEM127 is fundamentally sound. While the significance was deemed important, the strength of the evidence was found to be solid, although additional validation work would strengthen the findings.

  2. Reviewer #1 (Public Review):

    In this study, authors have investigated the effects of TMEM127 depletion on RET regulation and function that could potentially contribute to PCC pathogenesis. They have demonstrated that the loss of TMEM127 leads to cell surface accumulation and constitutive activation of RET due to membrane organization, leading to reduced efficiency of endocytosis, decreased internalization of RET, and a global impairment of membrane trafficking. TMEM127 depletion has contributed to increased RET half-life, constitutive RET-mediated signaling, increased membrane protein diffusibility, impaired normal membrane transitions, and inappropriate accumulation of actively signaling RET molecules at the cell membrane. Collectively, these findings have shown that the mis-localized RET is the pathogenic mechanism in TMEM127-mutant pheochromocytoma.

    Experimental design and mechanistic studies are thorough and sound. The methodological weakness lies in the lack of pheochromocytoma cell line utility to reproduce novel findings observed in generated cell lines. This may represent a significant challenge that could undermine the inferred value of these potentially paradigm-changing findings. 3-dimensional patient-derived pheochromocytoma organoid in vitro model and/or patient-derived organoid xenograft in vivo model may aid in reconciling these exciting new findings and factoring in that the pheochromocytoma is a hormonally active tumor.

    Fundamentally, the authors have successfully achieved all proposed aims supported by their conclusions.

    These findings carry potentially significant clinical impact and may offer new therapeutic venues in patients with pheochromocytoma.

  3. Reviewer #2 (Public Review):

    Summary: Walker et al have proposed that the tumor suppressor TMEM127 converges with RET activation to drive adrenal phenochromocytoma. RET is a common oncogene both in familial and sporadic forms of this cancer, and TMEM127 has also been observed as a loss of function mutation in sporadic disease. The authors hypothesize that loss of the TMEM127 might signal stabilization of RET on the cell surface, mimicking an activating mutation. Through a nice set of experiments, they show that TMEM127 loss impairs endosome function and promotes RET surface accumulation. This expression was resistant to GDNF, suggesting that recycling via endosome recirculation was impaired such that the half-life of RET on the cell surface was extended. RET interaction with clathrin-coated pits was also disrupted, as the CCPs themselves were significantly smaller, and plasma membrane organization was affected by the impaired endosome recycling. Notably, a number of proteins were found to be accumulating on the cell surface via the purported mechanism, EGFR, TFR1, N cadherin, integrin beta 3. The authors applied a RET inhibitor to cells, showing decreased cellular proliferation.

    Strengths: In summary, this is an interesting finding, that is preliminary in nature and is incompletely validated currently. It is certainly worth further investigation as a central feature linking TMEM127 mutations and pheochromocytoma through a common pathway of RET activation by fixing this factor in an active state on the cell surface.

    Weaknesses: Although this is a provocative finding, and the authors test the interaction in a number of ways, there are several factors that limit the enthusiasm for this work as currently presented. The work is limited to one isogenic cell line with limited validation.