SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression

Abstract

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Endogenous Retroviral Elements in Human Development and Central Nervous System Embryonal Tumors

“…We observed co-expression of OCT4 and HML-2 env in AT/RT cells, further cementing the connection between the stem-cell transcription factor and expression of HML-2 transcripts [30]. Similarly, we found that there is robust concomitant expression of the neural stem cell marker Nestin in cells expressing high HML-2 env protein in AT/RT patient-derived cell lines [30]. We also found that targeting HML-2 env expression in AT/RT resulted in significantly lower cell proliferation and disrupted the cell-cell communication required for cells to maintain pluripotency [30].…”

“…Thus, the regulation of HML-2 expression must be carefully regulated for cells to either retain stemness or differentiate and remain viable after differentiation. HML-2 env expression occurs in many tumor types and has been shown to imbue cancer cells with stem-cell-like characteristics [22,30,31]. We recently found high HML-2 env expression confers a stem-cell-like phenotype and contributes to active proliferation in atypical teratoid rhabdoid tumor (AT/RT) [30].…”

“…HML-2 env expression occurs in many tumor types and has been shown to imbue cancer cells with stem-cell-like characteristics [22,30,31]. We recently found high HML-2 env expression confers a stem-cell-like phenotype and contributes to active proliferation in atypical teratoid rhabdoid tumor (AT/RT) [30]. We observed co-expression of OCT4 and HML-2 env in AT/RT cells, further cementing the connection between the stem-cell transcription factor and expression of HML-2 transcripts [30].…”

“…We recently found high HML-2 env expression confers a stem-cell-like phenotype and contributes to active proliferation in atypical teratoid rhabdoid tumor (AT/RT) [30]. We observed co-expression of OCT4 and HML-2 env in AT/RT cells, further cementing the connection between the stem-cell transcription factor and expression of HML-2 transcripts [30]. Similarly, we found that there is robust concomitant expression of the neural stem cell marker Nestin in cells expressing high HML-2 env protein in AT/RT patient-derived cell lines [30].…”

“…Targeted HML-2 downregulation in metastatic breast cancer and in pancreatic cancer resulted in a concurrent decrease in NRAS protein suggesting HML-2 may target Ras genes [32,33]. When patient derived AT/RT cell lines were treated with shRNA targeting HML-2 env, a significant decrease in both env and N-RAS transcription occurred [30]. When the AT/RT cells were co-transfected with a CRISPRi construct (+gRNA targeting HML-2 LTR) and an N-RAS plasmid, HML-2 expression was higher by qRT-PCR compared with CRISPRi (+gRNA) and pcDNA CAT co-transfection, which served as a control [30].…”