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.

Figure 1

Immunostaining and Immunohistochemistry of patient-derived AT/RT cells and tissues. (a,b) Two resected patient AT/RT demonstrate strong immunostaining with HML-2 Envelope (Env) monoclonal antibody (brown). (c) Cerebrum from a normal brain is negative when stained with the same antibody. (d–l) AT/RT cell lines express markers of multiple stages of differentiation; representative images are shown. (d) CHLA 02, a patient derived AT/RT cell line, expresses Pax6 (red), a marker for neuroectoderm, merged with immunostaining for HML-2 Env (green). (e): This cell line also expresses Nestin (green), a marker of neural stem cells. (f) The same cell line expresses Oct4 (red), a pluripotency marker. (g,h) CHLA 04 cells, another AT/RT line, express Oct4 (green) and Nestin (red). (i) CHLA 05 cells also express Oct4 (green) and Nestin (red). (j) The same cell line also expresses βIII tubulin (red), a marker for neurons and HML-2 envelope (green). (k) CHLA 06 cells also express Nestin (red) and Oct4 (green). (l) The same cell line also expresses βIII tubulin (red) and HML-2 Env (green). Nuclei are stained with DAPI (blue). For IHC images, slides were scanned with a OptraScan Digital Pathology Scanner and processed in Adobe Photoshop for incorporation into the figure. For immunofluorescent images, an EVOS fluorescence microscope (AMG) was used. Images were acquired with the native software installed on the EVOS microscope and processed in Microsoft PowerPoint (https://www.thermofisher.com/us/en/home/technical-resources/software-downloads/evos-fl-cell-imaging-system.html).

Figure 2

Extracellular Vesicles are released from CHLA 02 and CHLA 04 cells and contain HML-2 Envelope (Env). (a,b) AT/RT cells release extracellular vesicles contain HML-2 Env. (a) Immunostaining CHLA 02 cells with HML-2 env polyclonal antibody (surface unit) (red) and CD98 (green), a cell-surface marker, shows Env is expressed in extracellular vesicles enclosed by the plasma membrane. Cell nuclei are stained with DAPI (blue); (b) CHLA 04 cells similarly immunostained; magnification is at 63X. (c) CHLA 02 cells incubated with secondary antibody only as a control for nonspecific immunostaining. (d,e) Electron microscopy shows extracellular vesicles forming on plasma membrane of CHLA 02 AT/RT cells (arrow heads). (f) Cell lysates from Tera, CHLA 02, and CHLA 04 cells express HML-2 env (left three lanes) while only purified extracellular vesicles from CHLA 02 and CHLA 04 express HML-2 env (three right lanes) on immunoblot (arrow). A fluorescent Zeiss confocal microscope (LSM510) was used to acquire images processed with Zeiss LSM 5 Image Browser and Microsoft PowerPoint for figure creation (https://www.embl.de/eamnet/html/downloads.html). The gel image has been cropped to focus on the bands of interest, the full gel image can be found in the supplementary material. Gel Image was taken with a FluorChem E system (https://www.proteinsimple.com/fluorchem.html) and images were processed with ImageJ and Microsoft PowerPoint for relative densitometry quantification and display. Electron microscopy images were taken with a JEOL 1200 EXII Transmission Electron Microscope (AMT digital camera system).

Figure 3

SMARCB1 regulates HERV-K (HML-2) env expression. (a) Restored SMARCB1 expression in CHLA 02 and CHLA 04 AT/RT cell lines results in downregulation of HML-2 transcription measured at 48 h by qRT-PCR. (b,c) SMARCB1 binds the HML-2 LTR significantly more than the promoter of control gene, HPRT. (b) SMARCB1 transfected 293 T cells show a significantly greater proportion of HML-2 LTR bound to SMARCB1 following immunoprecipitation compared to control, non-targeting IgG. (c) SMARCB1 transfected 293 T cells show no significant difference between non-targeting IgG bound to genomic HPRT and SMARCB1 bound to genomic HPRT. Percent input is a normalized value with input set to 100% (e.g. % input = 100*2^(input Ct − immunoprecipitated chromatin Ct). Ct is cycle threshold. (d,e) SMARCB1 knockdown results in increased transcription of HML-2 transcripts as measured with qRT-PCR. (d) HML-2 transcripts in 293 T cells transfected with scrambled shRNA control compared to shRNA targeting SMARCB1 at 24 h as measured by qRT-PCR. (e) HML-2 transcripts in neural stem cells transfected with scrambled shRNA control are significantly higher compared to transcripts in cells transfected with shRNA targeting SMARCB1 at 48 h. (qRT-PCR) Data was entered into Prism v9 for graph creation. [Error bars represent SEM. Statistics in Supplemental Table S6].

Figure 4

RNA sequencing of AT/RT cell lines. (a,b) HERV-K (HML-2) internal coding genes and LTR5_Hs are expressed from most chromosomes. (a) Table shows quantity of loci expressed on each chromosome (Chr). (b) Graphical depiction of HERV-K (HML-2) internal coding genes in (HERV-K-int) and LTR5_Hs in (long terminal repeat 5 human specific) expression in four AT/RT cell lines. The outer ring is comprised of the chromosomes from the human genome with the list of both “HERV-K-int” (internal coding sequence) and LTR5_Hs loci and black lines connecting them to their approximate location on the chromosome. The four rings in the center correspond to the CHLA 02, 04, 05, and 06 cell lines with the 02 cells being the outermost circle and the 06 cells the innermost circle. The segments of the central circles represent the chromosomes; blue and red lines correspond to the level of expression of either the LTR5_Hs or HERV-K (HML-2) labeled at that location (red denotes higher expression, blue denotes less). (c) Scheme for PCR amplification and Sanger sequencing validation of HML-2 transcripts. Shows a representation of primer position relative to potential env, rec, and np9 transcripts which are transcribed in the AT/RT cells. The forward primer used for amplifying env (1), rec (2), and np 9 (3) transcripts is positioned in the 5’ UTR while the reverse primer is positioned in the 3’ UTR. Because both primers are in the UTRs, env, rec and np9 transcripts can all be amplified with the same primer set. The image in (b) was generated with the RCircos package version 1.2.1 (https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-14-244).

Figure 5

Effect of the Downregulation of HML-2 on AT/RT. (a–d) A decrease in HML-2 expression leads to reduced cellular aggregates in CHLA 02 AT/RT cells. (a) HML-2 env transcript levels in CHLA 02 cells 48 h post transfection with either shRNA targeting env or CRISPRi targeting HML-2 LTR. (b) CHLA 02 cells transfected with CRISPRi + gRNA have reduced aggregate size following reduced expression of HML-2 mRNA at 24 h, 48 h, and 72 h post transfection. (c) CHLA 02 cells at 72 h post transfection with either pcDNA or (d) with CRISPRi + gRNA. (e–i) Downregulation of HML-2 results in AT/RT cell death and reduced cell aggregates. (e) CHLA 02 and CHLA 04 cells transfected with CRISPRi with or without gRNA targeting HML-2 LTR results in different proportions of viable cells at 96 h post transfection. (f) Percent of control (siRNA non-targeting) env transcription following transfection with siRNA targeting HML-2 env in CHLA 02 at 24 h. (g) CHLA 02 cell aggregates were reduced in size after 72 h following siRNA transfection targeting HML-2 env. (h) CHLA 02 at 72 h post transfection with both non-targeting siRNA and (i) cells treated with HML-2 env siRNA. (j–m) Lentiviral vectors with CRISPRi + gRNA targeting HML-2 and shRNA targeting HML-2 env result in decreased AT/RT viability and a reduction in HML-2 env protein expression. (j,k) CHLA 02 cytotoxicity at multiple time points post-transduction with lentivirus expressing either a CRISPRi construct with CRISPRi + gRNA targeting HERV-K (HML-2) LTR5_Hs or with CRISPRi without gRNA. (l) Percent viable cells 48 h post-transfection shRNA targeting HML-2 env. (m) env protein post-transfection as a percent of control transfection (either with CRISPRi and no gRNA or shRNA (−)). Graphs generated with Prism v9. [Error bars represent SEM. Statistics in Supplemental Table S6].

Figure 6

NRAS expression is downregulated post HML-2 downregulation, and its overexpression is sufficient to restore cellular proliferation to AT/RT cells. (a) Downregulation of HML-2 transcription with shRNA results in a decrease in NRAS expression. Transcription level of HML-2 env and NRAS measured with qPCR 48 h post transfection with shRNA targeting HML-2 env in CHLA 02. (b–g) Co-transfection with NRAS overexpression plasmid can overcome the effects of HML-2 downregulation. (b) Expression of env, gag, and NRAS transcripts 48 h post transfection in CHLA 04 cells with both CRISPRi + gRNA and NRAS plasmids. (c) Cell number in CHLA 04 AT/RT cells transfected with CRISPRi + gRNA and either a CAT plasmid or an NRAS construct and 5 days post-transfection. (d) Viable cell percentage 5 days post transfection with CRISPRi and either CAT or NRAS plasmids. Image of cells transfected with CRISPRi plus HML-2 gRNA and either CAT (e) or NRAS (f). (g) Quantification of cell aggregate size 5 days post-transfection with CRISPRi + gRNA and either CAT or NRAS plasmids. Graphs generated with Prism v9. [Error bars represent SEM. Statistics in Supplemental Table S6].

Figure 7

C-MYC binding to the HERV-K (HML-2) LTR. (a–d) C-MYC binds HML-2 LTR significantly less when SMARCB1 is expressed. Panel a: Relative quantification of HML-2 LTR bound to C-MYC transcription factor binding protein immunoprecipitated from either 293 T cells transfected with a SMARCB1 plasmid or a control (CAT) plasmid. (b) Relative quantification of housekeeping gene HPRT bound to C-MYC post-immunoprecipitation from 293 T cells transfected with either SMARCB1 or a control (CAT) plasmid. (c) Relative quantification of C-MYC bound to the HML-2 LTR post immunoprecipitation in CHLA 02 cells transfected with either SMARCB1 or with CAT plasmid. (d) Relative quantification of CMYC bound to housekeeping gene HPRT post immunoprecipitation in CHLA 02 cells transfected with either SMARCB1 or with CAT plasmid. Percent input is a normalized value with input set to 100% (e.g. % input = 100*2^(input Ct − immunoprecipitated chromatin Ct). Ct is cycle threshold. (e) C-MYC binds the HML-2 LTR in vitro. Biotinylated nucleotides with a non-targeting sequence, a scrambled canonical C-MYC binding sequence, or varying sequences from the Chr7p22.1a or Chr7p22.1b HML-2 LTR were incubated with recombinant C-MYC protein, washed, and run on an immunoblot to detect specific binding between C-MYC and different sequences. There is a white line between the the final LTR_939_944 and another ladder depicting that a portion of the gel was deleted to simplify the figure. The full gel image can be found in the supplemental information. (f) Contains a diagram of the mechanism by which HML-2 expression is regulated in the absence or presence of SMARCB1 protein. (f1) shows how HML-2 is actively expressed when C-MYC is bound to the LTR (promoter) when SMARCB1 protein is not expressed, while (f2a,b) depict the SMARCB1 mediated inhibition of C-MYC activation of HML-2 transcription. Graphs generated with Prism v9. (f) Generated in Microsoft PowerPoint. [Error bars represent SEM. Statistics in Supplemental Table S6].