LncRNA LITATS1 suppresses TGF-β-induced EMT and cancer cell plasticity by potentiating TβRI degradation

Abstract

Epithelial cells acquire mesenchymal phenotypes through epithelial-mesenchymal transition (EMT) during cancer progression. However, how epithelial cells retain their epithelial traits and prevent malignant transformation is not well understood. Here, we report that the long noncoding RNA LITATS1 (LINC01137, ZC3H12A-DT) is an epithelial gatekeeper in normal epithelial cells and inhibits EMT in breast and non-small cell lung cancer cells. Transcriptome analysis identified LITATS1 as a TGF-β target gene. LITATS1 expression is reduced in lung adenocarcinoma tissues compared with adjacent normal tissues and correlates with a favorable prognosis in breast and non-small cell lung cancer patients. LITATS1 depletion promotes TGF-β-induced EMT, migration, and extravasation in cancer cells. Unbiased pathway analysis demonstrated that LITATS1 knockdown potently and selectively potentiates TGF-β/SMAD signaling. Mechanistically, LITATS1 enhances the polyubiquitination and proteasomal degradation of TGF-β type I receptor (TβRI). LITATS1 interacts with TβRI and the E3 ligase SMURF2, promoting the cytoplasmic retention of SMURF2. Our findings highlight a protective function of LITATS1 in epithelial integrity maintenance through the attenuation of TGF-β/SMAD signaling and EMT.

Keywords: LINC01137; TGF-β type I receptor; ZC3H12A-DT; epithelial-mesenchymal transition; transforming growth factor-β.

Figure 1. LITATS1 is a TGF‐β‐induced lncRNA

1. Scheme for screening lncRNAs induced by TGF‐β. MCF10A‐M1, MCF10A‐M2, and MDA‐MB‐231 cells were treated without (0 h) or with TGF‐β for 2 h, 8 h, or 24 h. RNA samples (biological triplicates) were collected for RNA‐seq, and lncRNAs induced by TGF‐β were selected for further analysis.

2. Heatmap showing the log2 fold changes in the 15 lncRNA hits induced by TGF‐β at all three time points (2 h, 8 h, and 24 h vs. 0 h) in at least two cell lines.

3. LITATS1 expression upon SMAD4 knockdown (as detected by RT–qPCR) in MDA‐MB‐231 cells. Cells were serum starved for 16 h and TGF‐β was added for 4 h. Representative results from a minimum of three independent experiments are shown.

4. LITATS1 expression (as detected by RT–qPCR) in HEK293T cells. Cells were transfected without (Co.vec) or with the constitutively active TGF‐β type I receptor (caTβRI) ectopic expression construct. Representative results from a minimum of three independent experiments are shown.

5. Effect of TGF‐β on LITATS1 promoter activity as determined by luciferase reporter assays. HepG2 cells were transfected with empty pGL4 vector (Co.vec) or with two indicated LITATS1 promoter luciferase reporters (LITATS1‐P1 and LITATS1‐P2). Cells were stimulated with ligand buffer as the vehicle control (−), BMP6 (50 ng/ml), or TGF‐β (5 ng/ml) for 16 h. Representative results from a minimum of three independent experiments are shown.

6. Effect of caTβRI and SMAD3 on LITATS1 promoter activity as determined by luciferase reporter assays. HepG2 cells were transfected with ectopic expression constructs for the LITATS1 promoter 2 luciferase reporter (LITATS1‐P2) and caTβRI or SMAD3 and were then stimulated with or without TGF‐β for 16 h. Representative results from a minimum of three independent experiments are shown.

7. Schematic representation of the genomic location of LITATS1 and its neighboring genes. The arrows indicate the direction of transcription.

8. CPAT software was used to predict the coding potential of protein‐coding mRNAs (ACTB2 and GAPDH), well‐annotated lncRNAs (Xist and NKILA), and LITATS1.

9. Expression analysis of lncRNA H19, NEAT1, and LITATS1 expression levels in the cytoplasmic and nuclear fractions of MCF10A‐M1, MCF10A‐M2, and MDA‐MB‐231 cells. Representative results from a minimum of three independent experiments are shown.

10. RNA fluorescence in situ hybridization was performed to evaluate LITATS1 expression and subcellular localization in A549 cells. Cells were treated with or without TGF‐β for 2 h. Representative images are shown in the left panel, and signal quantification data are shown in the right panel. Scale bar = 10 μm. Representative results from two independent experiments are shown.

Data information: TGF‐β was applied at a final concentration of 5 ng/ml. (C, D, E, F) are expressed as the mean ± SD values from three biological replicates (n = 3). (J) is expressed as the mean ± SD values from 30 biological replicates (n = 30). *0.01 < P < 0.05; **0.001 < P < 0.01; ***0.0001 < P < 0.001; ****P < 0.0001; NS, not significant. Statistical analysis was based on the unpaired Student's t‐test. Source data are available online for this figure.

Figure EV1. LITATS1 is a direct target gene of TGF‐β, related to Fig 1

1. LITAS1 expression (as analyzed by RT–qPCR) in MDA‐MB‐231 (left) or A549 (right) cells upon TGF‐β stimulation for indicated durations. Representative results from two independent experiments are shown.

2. SMAD4 expression (as analyzed by RT–qPCR) in MDA‐MB‐231 cells upon shRNA‐mediated SMAD4 knockdown. Representative results from a minimum of three independent experiments are shown.

3. Analysis of LITATS1 promoter fragment‐mediated transcriptional activity in A549 cells. Cells were transfected with ectopic expression constructs for the LITATS1 promoter 2 luciferase reporter (LITATS1‐P2) and stimulated with or without TGF‐β for 16 h. Representative results from two independent experiments are shown.

4. Effect of caTβRI and SMAD3 on LITATS1 promoter activity as determined by luciferase reporter assays. HepG2 cells were transfected with ectopic expression constructs for the LITATS1 promoter 2 luciferase reporter (LITATS1‐P2) and caTβRI or SMAD3 and were then stimulated with or without TGF‐β for 16 h. Representative results from a minimum of three independent experiments are shown.

5. Analysis of LITATS1 promoter fragment‐mediated transcriptional activity in HepG2 cells. Cells were transfected with empty pGL4 vector (Co.vec) or expression constructs for the four indicated LITATS1 promoter luciferase reporters (P2–P5). Cells were stimulated with or without TGF‐β for 16 h. The fold changes between the −TGF‐β and +TGF‐β groups are indicated. Representative results from a minimum of three independent experiments are shown.

6. Schematic representation of wild‐type (WT) and mutant (MUT) LITATS1 promoter 6 (P6).

7. Schematic illustration of LITATS1 exons and introns, the target sites of primers for 5′‐ and 3′‐RACE, and the target sites of shRNAs for LITATS1 knockdown and gRNAs for CRISPRa‐mediated LITATS1 overexpression. TSS: transcription start site. The results of agarose gel electrophoresis of the LITATS1 5′‐ and 3′‐RACE DNA products are shown in the lower panel. Representative DNA gel images from two independent experiments are shown.

8. Subcellular distribution of LITATS1 expression (as detected by RNA fluorescence in situ hybridization) in A549 cells. Cells with or without stable LITATS1 knockdown were incubated in the presence or absence of TGF‐β for 2 h. Representative images are shown in the left panel, and signal quantification data are shown in the right panel. Scale bar = 10 μm. Representative results from two independent experiments are shown.

9. Lack of effect of TGF‐β on the subcellular distribution of LITATS1 in A549 cells. Quantification of the LITATS1 nuclear:cytoplasmic signal ratio for each cell was based on the RNA fluorescence in situ hybridization results shown in Fig 1J. Representative results from two independent experiments are shown.

Data information: TGF‐β was applied at a final concentration of 5 ng/ml. (A–E) are expressed as the mean ± SD values from three biological replicates (n = 3). (H, I) are expressed as the mean ± SD values from 30 biological replicates (n = 30). **0.001 < P < 0.01; ***0.0001 < P < 0.001; ****P < 0.0001; NS, not significant. Statistical analysis was based on the unpaired Student's t‐test. Source data are available online for this figure.

Figure 2. LITATS1 expression correlates with better prognosis in breast cancer and lung cancer patients

1. A

   LITATS1 expression in different breast cells as measured by RT–qPCR. Results from epithelial‐like and mesenchymal‐like cells are labeled in blue and green, respectively. Representative results from two independent experiments are shown.

2. B

   Comparison of LITATS1 expression in breast cancer classified by PAM50 subtypes.

3. C

   Quantification of LITATS1 expression levels by in situ hybridization in lung adenocarcinoma tissue microarrays. Representative images (bar = 100 μm) and zoomed images (bar = 20 μm) of in situ hybridization results in lung adenocarcinoma and matched adjacent normal tissues are shown in the left panel. The comparison of the LITATS1 staining index between the paired tissues is shown in the right panel. Tissue pairs with higher LITATS1 expression in the normal tissue (normal) than in the lung adenocarcinoma tissue (tumor) are highlighted in red, whereas tissue pairs with lower LITATS1 expression in the normal tissue than in the tumor tissue are highlighted in green.

4. D

   Kaplan–Meier survival curves of relapse‐free survival in 175 breast cancer patients stratified by LITATS1 expression. LITATS1 expression was measured by in situ hybridization in breast cancer tissue microarrays.

5. E–H

   Kaplan–Meier survival curves of overall survival (E), distant metastasis‐free survival (F), and relapse‐free survival (G) in breast cancer patients and overall survival (H) in non‐small cell lung cancer patients stratified by LITATS1 expression. The data were generated via Kaplan–Meier Plotter (https://kmplot.com/analysis/).

Data information: (A) is expressed as the mean ± SD values from three biological replicates (n = 3). (B) is represented as box‐and‐whisker plots with 5–95 percentile line representing the median of each group. Numbers below the plot represent patient numbers (biological replicates). (C) is expressed as the mean ± SD values from 49 biological replicates (n = 49). *0.01 < P < 0.05; **0.001 < P < 0.01; ****P < 0.0001. In (A, B), statistical analysis was based on the unpaired Student's t‐test. In (C), statistical analysis was based on the paired Student's t‐test. In (D–H), the log‐rank (Mantel‐Cox) test was applied to calculate the statistical significance. Source data are available online for this figure.

Figure 3. LITATS1 knockdown potentiates EMT, cell migration, and cell extravasation

1. A, B

   Effect of LITATS1 on TGF‐β‐induced EMT marker expression in MCF10A‐M2 upon CRISPRa‐mediated LITATS1 overexpression (A) or shRNA‐mediated knockdown (B). GAPDH or α/β‐Tubulin, loading control. The results of LITATS1 overexpression and knockdown are shown in Appendix Fig S4A and Fig EV2B. Representative results from a minimum of three independent experiments are shown.

2. C

   Immunofluorescence analysis of F‐actin expression and localization in A549 cells upon shRNA‐mediated LITATS1 depletion. Cells were treated with or without TGF‐β for 24 h. Nuclei were visualized by DAPI staining. Scale bar = 30 μm. The result of LITATS1 knockdown is shown in Appendix Fig S4C. Representative results from two independent experiments are shown.

3. D, E

   An IncuCyte chemotactic migration assay was performed to evaluate the effect of LITATS1 ectopic expression (D) or knockdown (E) on TGF‐β‐induced MDA‐MB‐231 cell migration. The results of LITATS1 overexpression and knockdown are shown in Appendix Fig S4D and E. Representative results from two independent experiments are shown.

4. F, G

   In vivo zebrafish extravasation experiments with MDA‐MB‐231 cells upon ectopic LITATS1 expression (F) or LITATS1 knockdown (G). Representative zoomed images of the tail fin area are shown in the left panels. Extravasated breast cancer cell clusters are indicated with yellow arrows. Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panels. Whole zebrafish image, bar = 309.4 μm; zoomed image, bar = 154.7 μm. Representative results from two independent experiments are shown.

Data information: TGF‐β was applied at a final concentration of 1 ng/ml. (D, E) are expressed as the mean ± SD values from four biological replicates (n = 4). (F, G) are expressed as the mean ± SD values from 30 biological replicates (n = 30). *0.01 < P < 0.05; ***0.0001 < P < 0.001; ****P < 0.0001. In (D, E), statistical analysis was based on two‐way ANOVA. In (F, G), statistical analysis was based on the unpaired Student's t‐test. Source data are available online for this figure.

Figure EV2. LITATS1 inhibits TGF‐β‐induced EMT and migration in A549 cells, related to Fig 3

1. Effect of LITATS1 on TGF‐β‐induced EMT marker expression in A549 cells upon ectopic LITATS1 expression. Cells were stimulated without or with different concentrations of TGF‐β for 2 or 5 days as indicated. GAPDH, loading control. The results of LITATS1 overexpression are shown in Appendix Fig S4B. Representative blots from two independent experiments are shown.

2. LITATS1 expression (as analyzed by RT–qPCR) in MCF10A‐M2 cells upon shRNA‐mediated knockdown. Representative results from a minimum of three independent experiments are shown.

3. GSEA of positive correlations between (manipulated) LITATS1 expression and the EMT gene signature.

4. An IncuCyte chemotactic migration assay was performed to evaluate the effect of LITATS1 ectopic expression on TGF‐β‐induced cell migration in A549 cells. Representative results from two independent experiments are shown.

5. Workflow of the breast cancer extravasation experiment in a zebrafish embryo xenograft model. Blood vessels and cancer cells are fluorescently labeled in green and red, respectively.

Data information: (B, D) are expressed as the mean ± SD values from three (n = 3) and six (n = 6) biological replicates, respectively. **0.001 < P < 0.01; ****P < 0.0001. In (B), statistical analysis was based on the unpaired Student's t‐test. In (D), statistical analysis was based on two‐way ANOVA. Source data are available online for this figure.

Figure 4. LITATS1 suppresses TGF‐β/SMAD signaling and EMT

1. GSEA of positive correlations between (manipulated) LITATS1 expression and the TGF‐β gene response signature.

2. Effect of LITATS1 misexpression on TGF‐β/SMAD3 transcriptional activity in HepG2 cells. Cells were transfected with expression constructs for the TGF‐β‐induced SMAD3/4‐dependent CAGA‐luc transcriptional reporter and the LITATS1 misexpression construct. The results of LITATS1 misexpression are shown in Fig EV3A. Representative results from a minimum of three independent experiments are shown.

3. Expression of TGF‐β target genes (as measured by RT–qPCR) in MDA‐MB‐231 cells without (Co.sh) or with (sh#1 and sh#2) LITATS1 depletion. Cells were serum starved for 16 h and treated with or without TGF‐β for 4 h. Representative results from a minimum of three independent experiments are shown.

4. Effect of LITATS1 knockdown on TGF‐β‐induced SMAD2 phosphorylation in MDA‐MB‐231 cells. Cells were serum starved for 16 h and stimulated with TGF‐β for the indicated durations. The p‐SMAD2 and total SMAD2 (t‐SMAD2) levels were analyzed by western blotting. GAPDH, loading control. Representative results from a minimum of three independent experiments are shown.

5. Effect of LITATS1 knockdown on E‐cadherin, N‐cadherin, and SNAIL expression in A549 cells. Cells were stimulated with vehicle control (−), SB431542 (SB; 10 μM), or TGF‐β (Τβ) for 24 h, and protein expression was analyzed by western blotting. α/β‐Tubulin, loading control. Representative results from a minimum of three independent experiments are shown.

6. Effect of LITATS1 depletion (using two independent shRNAs, i.e., shLITATS1 #1 and #2) on F‐actin expression and localization (as evaluated by immunofluorescence) in A549 cells. DAPI staining was performed to visualize nuclei. Cells were stimulated with or without SB431542 (SB; 10 μM) for 48 h. Scale bar = 30 μm. Representative results from two independent experiments are shown.

7. IncuCyte wound healing migration assays were performed to evaluate the effect of TGF‐β signaling inactivation on MDA‐MB‐231 cell migration mediated by LITATS1 knockdown. Cells were treated with or without SB431542 (SB; 10 μM) during the migration assays. Representative results from two independent experiments are shown.

8. In vivo zebrafish extravasation experiments with MDA‐MB‐231 cells upon LITATS1 knockdown and blockage of TGF‐β signaling. Representative zoomed images of the tail fin area are shown in the left panels. Extravasated breast cancer cell clusters are indicated with yellow arrows. Analysis of the extravasated cell cluster numbers in the indicated groups is shown in the right panel. Whole zebrafish image, bar = 618.8 μm; zoomed image, scale bar = 154.7 μm. Representative results from two independent experiments are shown.

Data information: TGF‐β was applied at a final concentration of 1 ng/ml. (B, C) are expressed as the mean ± SD values from three biological replicates (n = 3). (G) is expressed as the mean ± SD from seven biological replicates (n = 7). (H) is expressed as the mean ± SD values from 30 biological replicates (n = 30). *0.01 < P < 0.05; **0.001 < P < 0.01; ***0.0001 < P < 0.001; NS, not significant. In (B, C, H), statistical analysis was based on the unpaired Student's t‐test. In (G), statistical analysis was based on two‐way ANOVA followed by Tukey's multiple comparisons test. Source data are available online for this figure.

Figure EV3. LITATS1 suppresses TGF‐β/SMAD signaling, related to Fig 4

1. A

   Expression analysis of LITATS1 (as measured by RT–qPCR) in HepG2 cells upon LITATS1 overexpression mediated by CRISPRa (left) or LITATS1 knockdown (right). Cells were transfected with the indicated constructs for LITATS1 overexpression or depletion and were stimulated with or without TGF‐β for 16 h. Representative results from two independent experiments are shown.

2. B

   Effect of ectopic expression of wild‐type (WT) or mutant (MUT) LITATS1 on TGF‐β/SMAD3 transcriptional activity in HepG2 cells. Cells were transfected with constructs for expression of the TGF‐β‐induced SMAD3/4‐dependent CAGA‐luc transcriptional reporter and overexpression of WT or MUT LITATS1. Representative results from a minimum of three independent experiments are shown.

3. C

   Expression analysis of PAI‐1 and SNAIL (as measured by RT–qPCR) in MCF10A‐M2 cells upon LITATS1 depletion. Representative results from a minimum of three independent experiments are shown.

4. D

   Expression analysis of LITATS1, PAI‐1, SMAD7, and PTHRP in MDA‐MB‐231 cells upon ectopic LITATS1 expression. Representative results from a minimum of three independent experiments are shown.

5. E

   Expression analysis of LITATS1, PAI‐1, CTGF, and SMAD7 in MCF10A‐M2 cells upon CRISPRa‐mediated LITATS1 overexpression. Representative results from a minimum of three independent experiments are shown.

6. F–I

   Effect of LITATS1 misexpression on the p‐SMAD2 level in MCF10A‐M2 or MDA‐MB‐231 cells. The p‐SMAD2 and total SMAD2 (t‐SMAD2) levels were quantified by western blotting. Vinculin or GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.

7. J

   An IncuCyte chemotactic migration assay was performed to evaluate the effect of TGF‐β signaling inactivation on MCF10A‐M2 cell migration mediated by LITATS1 knockdown. Cells were treated with or without SB431542 (SB; 10 μM) during the migration assays. Representative results from two independent experiments are shown.

Data information: In (A–I), cells were stimulated with or without TGF‐β (1 ng/ml). (A–E) are expressed as the mean ± SD values from three biological replicates (n = 3). (J) is expressed as the mean ± SD values from 12 biological replicates (n = 12). *0.01 < P < 0.05; **0.001 < P < 0.01; ***0.0001 < P < 0.001. In (A–E), statistical analysis was based on the unpaired Student's t‐test. In (J), statistical analysis was based on two‐way ANOVA. Source data are available online for this figure.

Figure 5. LITATS1 promotes the ubiquitination and degradation of TβRI

1. A, B

   Effect of ectopic LITATS1 expression (A) or LITATS knockdown (B) on TβRI expression in MDA‐MB‐231 cells. Right panel: quantification of relative TβRI protein levels. Vinculin or GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.

2. C, D

   Analysis of TβRI protein stability (as measured by western blotting) in MDA‐MB‐231 cells with ectopic LITATS1 expression (C) or LITATS knockdown (D). Cells were treated with CHX (50 μg/ml) for the indicated durations. Quantification of the relative TβRI protein level is shown in the lower panels. GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.

3. E

   TβRI expression in MDA‐MB‐231 cells with ectopic LITATS1 expression in the absence or presence of lysosome or proteasome inhibitors. Cells were incubated with vehicle control DMSO (−), the lysosome inhibitor BafA1 (20 nM) or HCQ (20 μM), or the proteasome inhibitor MG132 (5 μM) for 8 h. Vinculin, loading control. Representative results from a minimum of three independent experiments are shown.

4. F

   Effect of LITATS1 on TβRI polyubiquitination. HEK293T cells were transfected with ectopic expression constructs for HA‐Ubiquitin (HA‐Ub), caTβRI‐FLAG, and/or LITATS1. TβRI polyubiquitination was analyzed by western blotting. Representative blots from a minimum of three independent experiments are shown.

Data information: (A, B) are expressed as the mean ± SD values from three biological replicates (n = 3). (C, D) are expressed as the mean ± SD values from four biological replicates (n = 4). *0.01 < P < 0.05; **0.001 < P < 0.01; ***0.0001 < P < 0.001. Statistical analysis was based on the unpaired Student's t‐test. Source data are available online for this figure.

Figure EV4. LITATS1 enhances TβRI degradation, related to Fig 5

1. Expression analysis of TβRI in MCF10A‐M2 cells upon CRISPRa‐induced LITATS1 overexpression. TβRI protein and TBRI mRNA expressions were evaluated by western blotting (left) and RT–qPCR (right), respectively. GAPDH, loading control. Representative results from a minimum of three independent experiments are shown.

2. Expression analysis of TβRI in HEK293T cells upon ectopic expression of LITATS1. Cells were transfected with expression constructs for caTβRI‐HA and/or LITATS1. Protein expression was evaluated by western blotting (left) and quantification of the relative TβRI protein level is shown in the right panel. GAPDH, loading control. Representative results from a minimum of three independent experiments are shown.

3. Expression analysis of TBRI mRNA in MDA‐MB‐231 cells upon LITATS1 ectopic expression (left) or LITATS1 knockdown (right). Representative results from a minimum of three independent experiments are shown.

4. Expression analysis of TβRI protein in A549 cells upon LITATS1 knockdown. Protein expression was evaluated by western blotting (left) and quantification of the relative TβRI protein level is shown in the right panel. GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.

5. Effect of CHX on TβRI expression (as measured by western blotting) in HEK293T cells transfected with expression constructs for caTβRI‐HA and/or LITATS1. Cells were treated with CHX (50 μg/ml) for the indicated durations. GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.

6. Analysis of TβRI expression (as measured by western blotting) in HEK293T cells transfected with expression constructs for caTβRI‐HA and/or LITATS1 in the presence or absence of the indicated chemical compounds. Cells were incubated with vehicle control (DMSO (−)), a proteasome inhibitor (MG132 (5 μM)), or a lysosome inhibitor (BafA1 (20 nM) or HCQ (20 μM)) for 8 h. Vinculin, loading control. Representative blots from a minimum of three independent experiments are shown.

7. Effect of LITATS1 and/or TBRI knockdown on E‐cadherin, N‐cadherin, Vimentin, SNAIL, and TβRI expression in A549 cells. Cells without (Co.sh) or with stable LITATS1 knockdown by two independent shRNAs (#1 and #2) were transduced with the TΒRI shRNA expression construct, and protein expression was analyzed by western blotting. GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.

Data information: (A–D) are expressed as the mean ± SD values from three biological replicates (n = 3). (E) is expressed as the mean ± SD values from four biological replicates (n = 4). *0.01 < P < 0.05; **0.001 < P < 0.01; ***0.0001 < P < 0.001; NS, not significant. Statistical analysis was based on the unpaired Student's t‐test. Source data are available online for this figure.

Figure 6. LITATS1 interacts with TβRI and SMURF2

1. Interactions between LITATS1 and TGF‐β/SMAD signaling components or modulators were analyzed by RIP. RT–qPCR was performed to detect LITATS1 expression in immunoprecipitates from HEK293T cells transfected with expression constructs for the indicated proteins. Representative results from two independent experiments are shown.

2. Interactions between LITATS1 and TβRI or SMURF2 in MDA‐MB‐231 cells were detected by the CARPID approach. Cells with stable expression of TurboID–dCasRx were transduced without (Co.) or with (#1 and #2) LITATS1 targeting gRNAs. Cells were stimulated with or without TGF‐β (2.5 ng/ml) for 2 h and were then stimulated with biotin (500 μM) for 30 min. Western blotting was performed to detect SMURF2 and TβRI expression in whole‐cell lysates (Input) and immunoprecipitates (IP). GAPDH and HA‐dCasRx expression levels were measured for equal loading of Input samples and as the negative control or positive control, respectively, for proximity biotinylation in immunoprecipitate (IP) samples. Representative results from a minimum of three independent experiments are shown.

3. Interactions between LITATS1 and TβRI (left) or SMURF2 (right) were analyzed by RIP. MDA‐MB‐231 cells were stimulated with or without TGF‐β (5 ng/ml) for 4 h before RIP. RT–qPCR was performed to detect LITATS1 expression in immunoprecipitates from MDA‐MB‐231 cells. IgG was included as the control for immunoprecipitation. Representative results from two independent experiments are shown.

4. Interactions between LITATS1 and caTβRI or SMURF2 were analyzed by RNA pull‐down. Biotinylated 25x poly(A), antisense LITATS1 (LITATS1‐AS), or LITATS1 was incubated with lysates from HEK293T cells transfected with the caTβRI‐HA or MYC‐SMURF2 expression construct. Western blot analysis was performed to detect HA or MYC expression in whole‐cell lysates (Input) and immunoprecipitates (IP). Representative blots from a minimum of three independent experiments are shown.

5. In vitro RNA pull‐down assays were performed to evaluate the interactions between LITATS1 and SMURF1/2. In vitro‐transcribed antisense LITATS1 (LITATS1‐AS) or LITATS1 (LITATS1‐S) was incubated with recombinant FLAG‐tagged SMURF1 or SMURF2 protein. Western blotting analysis was performed to evaluate FLAG expression in input and IP samples. The amounts of RNA probes used for RNA pull‐down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.

6. Quantification of TβRI‐SMURF2 PLA in A549 cells with or without LITATS1 knockdown were treated with or without TGF‐β (5 ng/ml) for 2 h. Representative images are shown in Fig EV5F.

7. Effect of LITATS1 overexpression on SMURF2‐mediated TβRI polyubiquitination. HEK293T cells were transfected with expression constructs for HA‐Ubiquitin (HA‐Ub) and caTβRI‐FLAG and ectopic expression constructs for SMURF2 and/or LITATS1. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.

8. Effect of SMURF2 knockdown on LITATS1‐mediated TβRI polyubiquitination. MDA‐MB‐231 cells with stable HA‐Ub expression were transduced with expression constructs for LITATS1 and/or two different SMURF2 shRNAs, as indicated. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.

Data information: (C) is expressed as the mean ± SD values from three (n = 3) biological replicates. (F) is expressed as the mean values from 15 (n = 15) biological replicates. **0.001 < P < 0.01; ***0.0001 < P < 0.001; ****P < 0.0001. Statistical analysis was based on the unpaired Student's t‐test. Source data are available online for this figure.

Figure EV5. LITATS1 interacts with TβRI and SMURF2, related to Fig 6

1. Expression analysis (by western blotting) of FLAG‐tagged proteins in HEK293T cells. Representative blots from two independent experiments are shown.

2. Scheme of the CARPID workflow. B: biotin; RBP: RNA‐binding protein.

3. Expression analysis of LITATS1 in MDA‐MB‐231 cells upon CasRx‐mediated LITATS1 knockdown. Stable cells were serum starved for 16 h and stimulated with or without TGF‐β (1 ng/ml) for 4 h. The RT–qPCR results are expressed as the mean ± SEM of technical triplicates.

4. Normalization of LIATS1 expression values in the RIP samples (Fig 6C) to that in the input samples (Fig EV5E). Results are expressed as the mean ± SD values from three biological replicates (n = 3). NS, not significant. Statistical analysis was based on the unpaired Student's t‐test. Representative results from two independent experiments are shown.

5. Expression analysis of LITATS1 in the input samples corresponding to Fig 6C. Results are expressed as the mean ± SD values from three biological replicates (n = 3). ****P < 0.0001. Statistical analysis was based on the unpaired Student's t‐test. Representative results from two independent experiments are shown.

6. The endogenous interaction between TβRI and SMURF2 was evaluated by PLA. A549 cells with or without LITATS1 knockdown were treated with or without TGF‐β (5 ng/ml) for 2 h. The red and blue dots indicate the TβRI‐SMURF2 interaction and the staining of nuclei by DAPI, respectively. Scale bar = 23.2 μm. Representative images from two independent experiments are shown.

Source data are available online for this figure.

Figure 7. LITATS1 retains SMURF2 in the cytoplasm

1. A

   An in vitro RNA pull‐down assay was performed to evaluate the interaction between LITATS1 truncation mutants and SMURF2. Recombinant FLAG‐SMURF2 protein was incubated with antisense LITATS1 (LITATS1‐AS), LITATS1 (LITATS1‐S), or LITATS1 truncation mutants (T1‐T4). Western blot analysis was performed to evaluate FLAG expression in immunoprecipitates (IP). The amounts of RNA probes used for RNA pull‐down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.

2. B

   Schematic representation of full‐length SMURF2 (FL) and the truncation mutants (T1‐T5) tested.

3. C, D

   RNA pull‐down assays were performed to evaluate the interaction between LITATS1 and full‐length SMURF2 or its truncation mutants (T1‐T5) expressed in HEK293T cells. Western blotting analysis was performed to evaluate FLAG expression in input and immunoprecipitate (IP) samples. Representative results from a minimum of three independent experiments are shown.

4. E

   SMURF2 expression and localization (as measured by immunofluorescence) upon LITATS1 depletion in A549 cells. DAPI staining was performed to visualize nuclei. Scale bar = 23.2 μm. Representative results from two independent experiments are shown.

5. F

   Effect of LITATS1 knockdown on SMURF2 localization in A549 cells. After subcellular protein fractionation, western blotting was performed to detect SMURF2 expression in whole‐cell lysates (Total) and the cytoplasmic (Cyto) and nuclear (Nuc) fractions. The levels of the cytoplasmic marker GAPDH and the nuclear marker Lamin A/C are included to demonstrate subcellular protein fractionation. Representative results from two independent experiments are shown.

6. G

   Schematic working model. TGF‐β‐induced LITATS1 interacts with TβRI and SMURF2 and potentiates cytoplasmic retention of SMURF2. The expression of LITATS1 potentiates TβRI polyubiquitination and proteasomal degradation, resulting in suppression of TGF‐β signaling, TGF‐β‐induced EMT, and cancer cell migration/invasion.

Source data are available online for this figure.