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. 2023 Jul 17:33:713-732.
doi: 10.1016/j.omtn.2023.07.008. eCollection 2023 Sep 12.

Overexpression of the mitochondrial anti-viral signaling protein, MAVS, in cancers is associated with cell survival and inflammation

Sweta Trishna  1 , Avia Lavon  1 , Anna Shteinfer-Kuzmine  2 , Avis Dafa-Berger  1 , Varda Shoshan-Barmatz  1   2
Affiliations

Overexpression of the mitochondrial anti-viral signaling protein, MAVS, in cancers is associated with cell survival and inflammation

Sweta Trishna et al. Mol Ther Nucleic Acids. .

Abstract

Mitochondrial anti-viral signaling protein (MAVS) plays an important role in host defense against viral infection via coordinating the activation of NF-κB and interferon regulatory factors. The mitochondrial-bound form of MAVS is essential for its anti-viral innate immunity. Recently, tumor cells were proposed to mimic a viral infection by activating RNA-sensing pattern recognition receptors. Here, we demonstrate that MAVS is overexpressed in a panel of viral non-infected cancer cell lines and patient-derived tumors, including lung, liver, bladder, and cervical cancers, and we studied its role in cancer. Silencing MAVS expression reduced cell proliferation and the expression and nuclear translocation of proteins associated with transcriptional regulation, inflammation, and immunity. MAVS depletion reduced expression of the inflammasome components and inhibited its activation/assembly. Moreover, MAVS directly interacts with the mitochondrial protein VDAC1, decreasing its conductance, and we identified the VDAC1 binding site in MAVS. Our findings suggest that MAVS depletion, by reducing cancer cell proliferation and inflammation, represents a new target for cancer therapy.

Keywords: MAVS; MT: Non-coding RNAs; VISA; cancer; cell proliferation; inflammation; mitochondria; siRNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

None
Graphical abstract
Figure 1
Figure 1
MAVS overexpression in diverse types of cancer (A–C) Formalin-fixed and paraffin-embedded US-Biomax tissue microarray slides containing the indicated tissues from cancer patients (n = 20) and normal tissue (n = 5) were immunohistochemistry stained using anti-MAVS antibodies and hematoxylin stained, as described in materials and methods. Representative images from sections of the different indicated tumors and corresponding healthy tissues are shown. The slides were incubated overnight at 4°C with anti-MAVS antibodies in PBS containing 1% BSA and then with secondary antibodies in PBS containing 1% BSA. The slides were subsequently treated with 3′3-diaminobenzidine tetra-hydrochloride (DAB) and counterstained with hematoxylin. Negative controls without primary antibody incubation were also performed. Sections of tissue were observed under an Leica microscope, and images were taken at 200× magnification with the same light intensity and exposure time. The percentage of the tumor sections stained at the intensity indicated on the scale above are presented. (D) Peripheral blood mononuclear cells (PBMCs) were obtained from chronic lymphocytic leukemia (CLL) patients (n = 16) and healthy donors (n = 13) using Ficoll-Paque PLUS (GE Healthcare, Israel) density gradient centrifugation, as described previously. Representative immunoblots of cell lysates of PBMCs derived from CLL patients and healthy donors subjected to SDS-PAGE and immunoblotting using anti-MAVS and anti-actin antibodies are shown. (E) Representative immunoblots using anti-MAVS antibodies of tissue lysates of lung cancer samples from tumor tissue (T, n = 22) and healthy tissue (H, n = 22), each derived from the same lung of a lung cancer patient. (F) Quantification of MAVS levels in CLL and lung cancer patients relative to healthy donors presented as fold change. The results are the mean ± SD. (G) HEK-293, Hela, SHSY-5Y, PC3, A549, MDA-MB-231, and UMUC3 cell lines were grown in the appropriate medium, and cell lysates (10 μg of protein) were subjected to SDS-PAGE and immunoblotting with anti-MAVS and citrate synthase (CS) antibodies (n = 3). (H) Quantitative analysis of MAVS and CS expression levels in the different cell lines were presented relative to the level in SHSY-5Y cells for MAVS and to A549 cells for CS. (I) T-REx-293 cells were transiently transfected with pcDNA3 or MAVS-pcDNA3 using metafectene as in the materials and methods section. MAVS expression was analyzed by immunoblotting 48 h post transfection. (J) Schematic presentation of the bicistronic nature of the MAVS transcript depicting met1 (methionine1) and met142 (methionine142) as the two translation initiation sites.
Figure 2
Figure 2
MAVS mitochondria localization, interaction with VDAC1, and identification of the interaction sequence (A) PC-3 cells were immunofluorescence stained for AIF-1 and MAVS (SC-166583) using specific antibodies, and the nucleus was stained with DAPI (blue). (B) Coomassie-stained, purified VDAC1 and MAVS. (C) Schematic presentation of the VDAC1 channel activity setup. (D) VDAC1 was reconstituted into a PLB and currents through VDAC1, in response to a voltage step from 0 to 10 mV or to 50 mV, were recorded before and 15 min after the addition of MAVS. (E) Multi-channel recordings of VDAC1 conductance as a function of voltage, and the average steady-state conductance of VDAC1 before (●) and 15 min after the addition of MAVS (o) (n = 3). (F) Fluorescently labeled, purified VDAC1 (138 nM) was incubated for 30 min at 37°C with MAVS (2.4–100 μM), and MST was performed, and the revealed Kd was obtained. (G,H) Proximity ligand assay (PLA) between MAVS and VDAC1 in PC-3 (control) and PC-3 MAVS-KO cells (G) and PLA quantification (H). (I) Immunoblotting analysis of MAVS (ab31334) expression levels in PC-3 cells expressing (control) and KO for MAVS. RU indicates relative units. (J) Schematic presentation of peptide arrays and detection of peptides interacting with VDAC1. (K) Glass-bound peptide array consisting of overlapping peptides derived from 19 VDAC1-interacting proteins including MAVS was incubated 4 h with purified VDAC1 (64 nM) and then blotted with anti-VDAC1 antibodies against an internal sequence or against the VDAC1-N terminus, followed by incubation with HRP-conjugated anti-mouse IgG and detection using a chemiluminescence kit. Dark spots represent binding of VDAC1 to peptides derived from VDAC1-interacting proteins. (L) Sequences of MAVS-derived peptides (MA-IL13 and MA-IL14) representing theVDAC1 interacting peptides (dark spots) with the red-colored peptides.
Figure 3
Figure 3
MAVS silencing inhibited cancer cell proliferation and nuclear localization of MAVS (A and B) Immunoblotting analysis of MAVS (ab31334) expression levels in PC-3 cells, transfected for 48 h with the indicated concentration of si-NT or si-MAVS (A), and MAVS level quantification (B). β-actin immunostaining was used as a loading control. (C and D) Cells were transfected with si-NT (75 nM) or si-MAVS (75 nM), and MAVS expression levels were analyzed at the indicated times post transfection. Immunoblot (C) and quantitative analysis of both MAVS variants (70 and 50 kDa) (D) are shown. (E) Cell proliferation of PC-3 cells transfected with si-NT (75 nM) or si-MAVS (75 nM) was assayed 24 to 120 h post transfection using the SRB cell proliferation assay, as described in the materials and methods section. Inhibition of cell proliferation by MAVS silencing is presented as a function of time post transfection. The dashed red line points to the maximal cell proliferation inhibition. (F) Representative IF co-staining of Ki-67 and MAVS (SC16583) in control and si-MAVS-treated PC-3 cells, with the nucleus stained with DAPI. (G) The percentage of cells with nuclear Ki-67 was determined over 100 cells of control and si-MAVS-treated cells. (H) Enlargement of the squared area in (F) showing MAVS co-localization with Ki-67 at the nucleus (see arrows). (I) MAVS nuclear localization cells were treated with poly(I:C) (1.5 μg, 8 h) and then subjected to cell fractionation to cytosolic and nuclear fractions using a nuclear/cytosol fractionation kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions. Following centrifugation (16,000 × g, 10 min), the supernatant (cytosolic fraction), and pellet (nuclear fraction) were subjected to immunoblotting for MAVS, lamin B1 (nuclear), and GAPDH (cytosolic). Results are the means ± SEM (n = 3). ∗∗∗∗p ≤ 0.0001.
Figure 4
Figure 4
si-MAVS cell treatment inhibited poly(I:C) activation of IRF3 and reduced IFN-β expression (A) Representative IF staining of PC-3 cells treated with poly(I:C) for 6 and 12 h and IF co-stained for activated IRF3 (pIRF3) and MAVS (SC166583). Nuclei were stained with DAPI. (B) Analysis of nuclear pIRF3 levels in control and cells treated with poly(I:C) (1 or 2 μM) for the indicated time. (C) IF staining for pIRF3 in control and si-MAVS (75 nM)-treated PC-3 cells, with and without poly(I:C) (2 μM, 8 h) treatment. (D) Quantitative analysis of pIRF3 expression levels (□) and its nuclear localization (■). (E) pIRF3 expression levels were analyzed by immunoblotting in cells treated with si-NT or si-MAVS and with and without poly(I:C) treatment. RU indicates relative units. (F) Representative IF co-staining for IFN-β and MAVS (SC166583) in PC-3 cells treated with si-MAVS or si-NT followed by poly(I:C) treatment (1.5 μM, 8 h), as indicated. Nuclei were stained with DAPI. (G) Quantitative analysis of IFN-β and MAVS expression levels in si-NT- or si-MAVS-treated cells with and without poly(I:C) treatment. Results are the means ± SEM (n = 3). ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.
Figure 5
Figure 5
NF-κB activation by poly(I:C) is inhibited in si-MAVS-treated cells (A) PC-3 cells were treated with poly(I:C) for the indicated time and -IF co-stained with anti-p-NF-κB-p65 and anti-MAVS (SC166583), and the nucleus was stained with DAPI , and representative images are shown. (B) The percentage of cells with nuclear NF-κB-p65. (C–E) IF staining of PC-3 cells from control and si-MAVS-treated cells, with and without poly(I:C) treatment for p-NF-κB-p65 and MAVS, and the nucleus was stained with DAPI (C). The relative expressions of MAVS and NF-κB-p65 were analyzed (D). The percentage of cells with nuclear p-NF-κB-p65 (E). (F) MAVS (ab31334) levels in PC-3 cells transfected with si-NT (75 nM) or si-MAVS (75 nM) and treated with or without poly(I:C) were analyzed using immunoblotting. Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.
Figure 6
Figure 6
si-MAVS treatment reduced the poly(I:C)-induced ASC and NLRP3 expression and subsequent expression level of pro-IL-1β (A and B) Representative IF staining with anti-ASC and anti-NLRP3 antibodies of control and si-MAVS-treated PC-3 cells, with and without poly(I:C)-treatment (A) and quantitative analysis of NLRP3 and ASC expression levels (B). (C and D) Proximity ligand assay (PLA) in poly(I:C)-treated PC-3 cells also treated with si-NT or si-MAVS, showing increased direct interaction between NLRP3 and ASC in PC-3 cells treated with poly(I:C) and its inhibition in low MAVS-expressing cells (C). Quantitative analysis presented as the means ± SEM (n = 3) of the PLA product (D). (E–G) Representative IF co-staining of PC-3 cells for IL-1β and caspase-1 in cells treated subjected to si-NT (75 nM) or si-MAVS (75 nM) treatment and to poly(I:C), as indicated (E), and quantitative analysis of IL-1β and caspase-1 expression levels (F and G). (H) Immunoblot analysis of pro-IL-1β expression levels in poly(I:C)-treated cells with and without si-MAVS (75 nM) treatment. Actin immunostaining served as a loading control. (I) Caspase-1 activity was assayed in si-NT- (75 nM) or si-MAVS-treated (75 nM) cells as indicated, subjected to poly(I:C) treatment, using a caspase-1 colorimetric assay kit (BioVision, Milpitas, CA, USA). (J) IL-1β expression level was assayed in si-NT- (75 nM) or si-MAVS-treated (75 nM) cells as indicated, using an IL-1β immunoassay kit (Promega, Madison, WI, USA). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.
Figure 7
Figure 7
YAP and TAZ nuclear localization is inhibited by cell treatment with si-MAVS (A) Representative IF staining of PC-3 cells treated with 75 μM si-NT or si-MAVS, co-stained for YAP and F-actin (phalloidin). Yellow and white arrows point to YAP location in the nucleus in si-NT-treated cells and in the cytoplasm in si-MAVS-treated cells, respectively. (B) Percentage of cells with nuclear YAP in si-NT- and si-MAVS-treated cells, analyzed over 100 cells each. (C and D) Control and si-MAVS-treated cells were subjected to cytosolic and nuclear fractionation using a nuclear/cytosol fractionation kit (Biovision, Milpitas, CA) according to the manufacturer’s instructions. Following centrifugation (16,000 × g, 10 min), the supernatant (cytosolic fraction) and pellet (nuclear fraction) re-suspended to the original volume were subjected to immunoblotting for YAP, TAZ, GAPDH, and lamin B1 (C) and further quantified for YAP (D) and TAZ (F) levels in each fraction. The level of the protein in each fraction for si-MAVS-treated cells was calculated relative to its level in the control cells. (F–H) Representative IF staining of PC-3 cells treated with si-NT or si-MAVS, immunestained for TAZ and nucleus with DAPI (E), and quantification of TAZ expression levels (G) and of its nuclear and cytoplasmic localization (H). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.
Figure 8
Figure 8
Molecular mechanisms proposed for decreasing overexpressed MAVS levels in cancer cells inhibiting NLRP3 inflammasome formation, type-1 interferon signaling, and inflammation Retinoic acid inducible gene-I (RIG-I) served as cytosolic sensor for viral single-stranded ssRNA or endogenous RNA, used here as synthetic poly(I:C) and, by their binding, initiates anti-viral and inflammatory cell responses involving activation of MAVS to form a prion-like structure. This activated MAVS can regulate inflammation via three possible pathways. (1) Regulating type-1 interferon production (green arrows): MAVS activating TBK1, which phosphorylates interferon regulatory factor-3 (IRF3). Phosphorylated IRF3 forms a homodimer and enters the nucleus to turn on transcription of IFN-β and other inflammatory cytokines. This activation and nuclear translocation of pIRF3 is inhibited upon MAVS depletion. (2) Regulating the NF-kB-inflammation pathway (yellow arrows): activated MAVS recruits TRAF6, leading to activation of the NEMO and the IKK complex (containing Ikkα/β and NF-κBp65/p50), resulting in dissociation of the complex and activation of NF-κBp65/p50. The NF-κBp65/p50 translocates into the nucleus and upregulates transcription of NLRP3, pro-IL-6, and pro-IL-1β, leading to priming of NLRP3-induced inflammation. Upon poly(I:C) treatment, the NF-κBp65 expression increased with nuclear translocation, and both were decreased in si-MAVS-treated cells. (3) Activation of NLRP3 inflammasome assembly (blue arrows): activated MAVS recruits NLRP3 to the mitochondria and allows the assembly of the inflammasome following recruitment of ASC and caspase-1, converting the pro-IL-6 and pro-Il-1β to active IL-6 and Il-1β, promoting inflammation. MAVS is required to activate ASC for the inflammasome formation., Depleting MAVS in the cancer cells by si-MAVS reduced the expression of NRLP3 and ASC, thereby inhibiting NLRP3-associated processes such as formation of IL-6 and IL-1β.
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