Research Article: CFTR functions as a tumor suppressor in adenoid cystic carcinoma and its silencing reveals an associated vulnerability involving the Hsp70 chaperone system

Abstract:
Adenoid cystic carcinoma is a rare salivary gland malignancy of the head and neck region characterized by perineural invasion, distant metastasis, and a lack of effective targeted therapies. The molecular mechanisms underlying its progression remain poorly understood. In this study, we identified the cystic fibrosis transmembrane conductance regulator (CFTR) as a consistently downregulated gene in adenoid cystic carcinoma through differential expression analysis of multiple independent transcriptomic cohorts. Functional experiments were performed in SACC-83 and SACC-LM cell lines, including ectopic expression and knockdown of CFTR, RNA sequencing, quantitative polymerase chain reaction, and Western blot analyses. Pharmacological inhibition of Hsp70 using VER155008 and activation of the heat shock response by HSF1A were also assessed for effects on cell viability, migration, invasion, and apoptosis. Subcutaneous xenograft experiments in nude mice were conducted to evaluate tumor growth following stable CFTR knockdown in SACC-LM cells. Preliminary Kaplan-Meier survival analysis demonstrated that low CFTR expression was significantly associated with inferior overall survival, although this finding requires validation in a larger independent cohort. Protein interaction network modeling revealed that CFTR occupies a hub position within a conserved interaction network linking ion channel regulation, protein quality control, and kinase signaling. Functional experiments showed that ectopic expression of CFTR suppressed cell proliferation, migration, and invasion, whereas CFTR knockdown enhanced these malignant phenotypes, supporting a tumor-suppressive role for CFTR in these model systems. RNA sequencing of CFTR-overexpressing cells revealed coordinated downregulation of heat shock protein family members, including HSPA1A, HSPA1B, and HSPA6, with enrichment of pathways related to protein refolding. Quantitative polymerase chain reaction and Western blot analyses confirmed that CFTR expression is inversely associated with Hsp70 family members and MAPK1 (ERK2) at both mRNA and protein levels. Pharmacological inhibition of Hsp70 using VER155008 suppressed cell viability, migration, and invasion in a dose-dependent manner and induced apoptosis, phenocopying the effects of CFTR restoration. Subcutaneous xenograft experiments in nude mice further demonstrated that stable CFTR knockdown in SACC-LM cells markedly accelerated tumor growth. Conversely, activation of the heat shock response by HSF1A promoted proliferation, migration, and invasion, recapitulating the consequences of CFTR loss. Notably, Hsp70 inhibition was accompanied by compensatory upregulation of MAPK1 transcripts, suggesting that CFTR is inversely associated with Hsp70 and MAPK1 through parallel rather than linear mechanisms. These findings indicate that CFTR silencing in adenoid cystic carcinoma is accompanied by elevated Hsp70 chaperone expression, and that pharmacological targeting of Hsp70 phenocopies, rather than necessarily mediates, the tumor-suppressive effects of CFTR restoration in the SACC-83 and SACC-LM lineage. Targeting Hsp70 therefore provides a preclinical rationale, rather than direct translational evidence, for further investigation in this otherwise treatment-refractory malignancy.

Introduction:
Adenoid cystic carcinoma is a rare salivary gland malignancy of the head and neck region characterized by perineural invasion, distant metastasis, and a lack of effective targeted therapies. The molecular mechanisms underlying its progression remain poorly understood.

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