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Sources of Funding: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T32GM088129. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. No conflict of Interests exist for the authors.

Introduction

Despite advancements in surgical technique and targeted therapy, metastatic renal cell carcinoma (RCC) remains deadly with a <15% five-year survival. Understanding the molecular machinery driving the transition from localized malignancy to invasion/metastasis is critical if we are to improve patient outcomes. We hypothesize that loss of function of the tumor suppressor MERLIN (encoded by the neurofibromatosis type 2 gene, NF2) may be a key player in this transition. We set out to determine (1) the association between patient outcomes and NF2 mutation status, (2) MERLIN function in established RCC cell lines derived from primary and metastatic sites, and (3) to correlate MERLIN activity to in vitro measures of invasiveness and metastatic potential.

Methods

Utilizing publicly available tumor sequencing datasets, we determined the clinical association between NF2 mutations and tumor stage and clinical outcome. We characterized mRNA expression of NF2 and protein levels of MERLIN by quantitative PCR and Western immunoblotting in metastatic-derived (Caki1, ACHN) and non-metastatic-derived (786-O, A498, Caki2) RCC cell lines. Potential for invasion and metastasis were assayed in vitro using transwell invasion and colony forming assays, and in vivo by mouse xenograft (NU/J). The relative expression of MERLIN was assessed in a tissue microarray containing 31 primary tumors, 8 paired metastatic tumors, and 8 paired normal kidney samples by immunohistochemistry.

Results

NF2 mutations are enriched in higher grade (>pT3, pT4) and metastatic tumors compared to low grade or non-metastatic tumors (20-30% vs. 1.5%), and are associated with a decreased disease-free survival. Cell lines derived from metastatic RCC have lower NF2 expression than cell lines derived from local tumors, more importantly, MERLIN protein levels are decreased or undetectable by Western blot or IHC in metastatic derived cell lines. The metastatic lines Caki1 and ACHN have increased metastatic potential as measured by increased colony formation. MERLIN was absent in a tumor with sarcomatoid differentiation but was present in all 8 metastatic samples.

Conclusions

These results support a potential intriguing role for MERLIN loss of function in the clinically-relevant phenotypic transition from localized to invasive RCC. The model system described here will provide a crucial framework for testing MERLIN&[prime]s influence on invasiveness through knockdown and rescue of MERLIN activity in appropriate RCC cell lines.

Funding

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T32GM088129. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. No conflict of Interests exist for the authors.