AKT-induced lncRNA VAL promotes EMT-independent metastasis through diminishing Trim16-dependent Vimentin degradation

Despite the importance of AKT overactivation in tumor progression, results from clinical trials of various AKT inhibitors remain suboptimal, suggesting that AKT-driven tumor metastasis needs to be further understood. Herein, based on long non-coding RNA (lncRNA) profiling induced by active AKT, we identify that VAL (Vimentin associated lncRNA, LINC01546), which is directly induced by AKT/STAT3 signaling, functions as a potent pro-metastatic molecule and is essential for active AKT-induced tumor invasion, metastasis and anoikis resistance in lung adenocarcinoma (LAD). Impressively, chemosynthetic siRNAs against VAL shows great therapeutic potential in AKT overactivation-driven metastasis. Interestingly, similar to activated AKT in LAD cells, although unable to induce epithelial-mesenchymal transition (EMT), VAL exerts potent pro-invasive and pro-metastatic effects through directly binding to Vimentin and competitively abrogating Trim16-depedent Vimentin polyubiquitination and degradation. Taken together, our study provides an interesting demonstration of a lncRNA-mediated mechanism for active AKT-driven EMT-independent LAD metastasis and indicates the great potential of targeting VAL or Vimentin stability as a therapeutic approach.

substitution frequency (CSF) scores of VAL, HOTAIR, EGFR and TP53. Error bars represent the means ± SD of the PhyloCSF scores of three truncated RNA sequences of indicated mRNAs. f WB analysis of the coding potency of VAL when full length of VAL was cloned into pcDNA3.4 with a transcription initiating codon ATG and a C-terminal HA tag. Anti-HA antibody was used to probe transcribed proteins. Histone H3 with HA tag served as a positive control. g QRT-PCR analysis of the relative VAL levels in a panel of LAD cell lines as compared to the immortalized human bronchial epithelial cell line Beas2B. The representative images of three independent reproducible experiments are shown (b-d, f). Error bars represent the means ± SD derived from three independent experiments. Source data are provided as a Source data file.

Supplementary Figure 3. VAL promotes local invasion and intra-pulmonary and distant metastasis without inducing EMT. a QRT-PCR analysis of VAL levels in vector-control and VAL-overexpressing LAD cells. b
Representative bright-field morphology images in 5 random fields of vector-control or VALoverexpressing A549 and HCC827 cells. c QRT-PCR analysis shows that EMT-related cellular markers Kaplan-Meier analysis (Log-rank test) of the metastasis-free survival and overall survival of mice intracardially injected with the indicated cells. k-m VAL-overexpressed or -silenced LAD cells or corresponding vector-control cells labeled with luciferase expression were orthotopically implanted into the lung of nude mice (n = 5 per group). Representative bioluminescent images of pulmonary metastasis and quantitation of bioluminescent intensities analyzed by ROI tools are shown. Lung metastases were histologically confirmed by H&E staining. Scale bar: 30 μm (b, e), 100 μm (m).
Error bars represent the means ± SD derived from three independent experiments. Statistical analyses were performed by two-tailed unpaired Student's t test (a, c, d, f-i, l). n.s., not significant.
Source data are provided as a Source data file. Representative bioluminescent images of pulmonary metastasis, quantitation of bioluminescent intensities analyzed by ROI tools and H&E staining of lung tissue are shown. Scale bar: 100 μm.
Scatter dot plot represent the means ± SD. Statistical analyses were performed by two-way ANOVA multiple comparison analysis (b-d) and two-tailed unpaired Student's t test (a, f, g). n.s., not significant. Source data are provided as a Source data file. Source data are provided as a Source data file. dissolved in ddH2O (2 mg/ml) and added to culture medium at final concentration of 2 µg/ml in order to induce overexpression or silencing of the abovementioned genes. Transfection of plasmids or RNA oligonucleotides were performed using Lipofectamine 3000 reagent (Invitrogen) for luciferase reporter assays and molecular assays.

5' and 3' rapid amplification of cDNA ends (RACE) assays. 5'-and 3'-RACE assays were performed
to reveal the transcriptional initiation and termination sites of VAL using SMARTer RACE 5'/3' Kit (Takara, Tokyo, Japan) as instructed. Briefly, total RNAs were extracted from A549 and HCC827 cells using the Trizol reagent and treated with DNase I (Invitrogen) for first-strand cDNA synthesis, followed by 5'-RACE and 3'-RACE PCRs using multiple primers. PCR fragments were purified and cloned into the pMD19-T vector (Takara) and the insert with correct sequences as analyzed by sequencing was then subcloned into the expression vector plasmids.   (3, 3'-diaminobenzidine). The staining scores were determined based on both the intensity and proportion of positive cells in 10 random fields. Scores representing the proportion of positively stained tumor cells in sections was graded as follows: 0, no positive cells; 1, <10%; 2, 10%-50%; and 3, >50%. The staining intensity was recorded as 0 (no staining), 1 (week staining), 2 (moderate staining), and 3 (strong staining). The staining index (SI) was calculated as follows: SI=staining intensity × proportion of positively stained cells, resulting in scores of 0, 1, 2, 3, 4, 6, or 9. The SI scores were compared between LADs and corresponding adjacent non-cancerous lung specimens by paired t-test.
Single-molecule RNA fluorescence in situ hybridization (smFISH). Staining of VAL in cultured cells was performed by smFISH according to the Sanjay Tyagi lab's procedures 1 . Short probes labeled with Alexa Fluor 647 in 3' ends were designed in an online program (https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer) and synthesized by Invitrogen. Cells seeded on cover-slips in 24-well plates were briefly rinsed with PBS, fixed with 3.7% formaldehyde (pH 7.4) for 10 min at room temperature, permeabilized in 1 ml 70% ethanol for at least 1 h at 4°C and washed in hybridization buffer (100 mg/mL dextran sulfate and 10% formamide in 2× SSC) for 5 min. Subsequently, hybridization was carried out using anti-VAL oligonucleotide probe sets (125 nM in 50 μl hybridization buffer) for at least 4 h at 37°C in a moist chamber, followed by wash of the cells for 30 min at 37°C in wash buffer (10% formamide in 2× SSC). After the hybridization, cells on cover-slips were counterstained with 4', 6-diamidino-2phenylindole (DAPI), and representative images were obtained with a LSM810 confocal microscope using ZEN 2012 software version 8.1 (Carl Zeiss, Oberkochen, Germany).