2.1. LIX1L expression is positively correlated with mesenchymal markers, lymphatic metastasis, and high TNM stage in NSCLC tumor specimens.
To investigate the correlation of LIX1L expression with EMT markers in clinical tumor specimens, we detected LIX1L, E-cadherin, and Vimentin expressions in 81 human NSCLC tissues by IHC assay. LIX1L expression was observed in the cytoplasm and nucleus of tumor cells (Fig. 1A). The positive rate of LIX1L expression was 51.90% (42/81) and positively associated with lymphatic metastasis and high TNM stage (Table S1, P < 0.0001). E-cadherin expression was mainly located in the membrane of tumor cells (Fig. 1A) and the positive rate was 34.6% (28/81). E-cadherin expression is negatively associated with lymph node metastasis (Table S1, P = 0.0361) and TNM stage (P = 0.0247). Cytoplasm expression rate of Vimentin was 50.6% (41/81) and significantly correlated with lymph node metastasis (Fig. 1A, Table S1, P = 0.001). In addition, LIX1L expression was positively correlated with Vimentin expression (Fig. 1B; lung squamous cell carcinomas (LUAD), r = 0.483; lung adenocarcinomas (LUSC), r = 0.687; P < 0.05), while negatively correlated with E-cadherin expression (LUAD, r=-0.357; LUSC, r=-0.339; P < 0.05) in 41 LUAD and 40 LUSC specimens, respectively.
Next, we investigated the relationship between LIX1L and EMT markers at mRNA level in TCGA database. In 574 cases of LUAD, the correlation between LIXIL mRNA and CDH1 is low (Fig. 1C, r=-0.1037, P = 0.0129), while LIX1L is positively correlated with VIM (r = 0.4427, P < 0.0001) and ZEB1 (r = 0.5178, P < 0.0001). In 548 cases of LUSC, LIXIL expression is negatively correlated with CDH1 (Fig. 1C, r=-0.3245, P < 0.0001), and positively correlated with VIM (r = 0.6395, P < 0.0001) and ZEB1 (r = 0.5768, P < 0.0001). The above results indicated that LIX1L expression is positively correlated with mesenchymal markers at both mRNA and protein levels in NSCLC specimens.
2.2. LIX1L expression is significantly elevated in TGFβ1-treated EMT NSCLC cells.
Next, we treated H358, HCC827, and H1975 cells with TGFβ1 for 21d to induce EMT [28] and observed LIX1L expression in the cells. After TGFβ1 treatment, the cell morphology changed from cobblestone-like to elongated (Fig. 2A; Fig.S1A), accompanied by reduced E-cadherin expression and increased Vimentin expression (Fig. 2B, C; Fig.S1B, C). The results indicated that the TGFβ1-treated cells had undergone EMT (referred to as TGFβ1 cells thereafter). Compared with parental cells, the expression of LIX1L in TGFβ1 cells was significantly increased at both mRNA and protein levels (Fig. 2D, E; Fig.S1D, E). Besides, the microarray datasets (GSE49644) from GEO confirmed that LIX1L mRNA expression was increased in H358 (Log FC = 2.977, P < 0.0001) and HCC827 (Log FC = 2.852, P < 0.0001) cells treated with TGFβ1 for 48h (Fig. 2F). Taken together, the results from NSCLC tissues and cell lines indicated that LIX1L is a mesenchymal gene and might be functionally involved in the process of EMT.
2.3. Upregulated LIX1L expression promotes core EMT functions of NSCLC cells.
To investigate the biological functions of LIX1L during EMT, we knocked down LIX1L expression in H358-TGFβ1, HCC827-TGFβ1 (Fig. 3A), and H1975-TGFβ1 (Fig.S2A) cells using specific siRNA transfection in vitro. Transwells assay indicated that migration and invasion abilities were significantly decreased after LIX1L knockdown (Fig. 3B; Fig.S2B). CCK8 and clone formation assays showed that LIX1L knockdown significantly reduced cell proliferation (Fig. 3C, D; Fig.S2C, D). Resistance to anoikis is an important characteristic of EMT and a critical contributor to tumor invasion and metastasis [29, 30]. Next, we induced cell anoikis by culturing the cells in Poly-HEMA coated plates [31, 32] and detected the apoptosis using Western blot and flow cytometry (FCM). Western blot showed that the active caspase-3 protein level in LIX1L knockdown cells was increased compared with control cells (Fig. 3E; Fig.S2E). FCM (PE Annexin V-7AAD apoptotic assay) indicated that the apoptotic (anoikis) rate was significantly increased after LIX1L knockdown in TGFβ1 cells (Fig. 3F; Fig.S2F). In addition, Western blot showed that E-cadherin expression was obviously induced, while Vimentin and Twist1 protein expressions were inhibited upon LIX1L knockdown (Fig. 3G; Fig.S2G). On the contrary, LIX1L overexpression in H358, HCC827, and H1975 cells (Fig. 3H; Fig.S2H) increased migration, invasion (Fig. 3I; Fig.S2I), proliferation (Fig. 3J; Fig.S2J), colony formation (Fig. 3K; Fig.S2K), and anoikis resistance (Fig. 3L; Fig.S2L) abilities. Besides, LIX1L overexpression inhibited E-cadherin protein expression and increased Vimentin and Twist1 protein expressions (Fig. 3M; Fig.S2M). Together, gain or loss of function assays indicated that LIX1L expression plays important functions in the EMT process of NSCLC cells.
2.4. LIX1L is localized in the nucleoli and impacts ribosomal RNA (rRNA) synthesis in TGFβ1-treated EMT NSCLC cells
In NSCLC specimens, we have detected that positive LIX1L expression is distributed in the cytoplasm and nucleus of tumor cells. Next, we determined the localization of LIX1L in EMT NSCLC cells using confocal immunofluorescent (IF). Interestingly, we found that LIX1L protein is enriched in the nucleus, especially in the nucleoli of TGFβ1 cells (Fig. 4A; Fig.S3A) compared with parental cells. Considering that rRNA synthesis and ribosome biogenesis occurs in nucleoli, we set out to investigate whether ribosome biogenesis was influenced by LIX1L expression during EMT. RT-qPCR results showed that 47s and 45s pre-rRNA (indicators of rRNA synthesis [33–35]) levels were significantly increased in TGFβ1 cells (Fig. 4B, C; Fig.S3B, C) compared with parental cells. Besides, H&E staining showed that the nucleolar size was increased (reflecting upregulation of ribosome biogenesis [25]) in TGFβ1 cells (Fig. 4D; Fig.S3D). LIX1L knockdown led to a significant decrease in 47s and 45s pre-rRNA expressions and nucleolus size (Fig. 4E-G; Fig.S3E-G). In contrast, overexpression of LIX1L significantly induced 47s and 45s pre-rRNA expressions in H358, HCC827 (Fig. 4H, I), and H1975 cells (Fig.S3H, I).
Next, we explored the effect of LIX1L on the expression of several key ribosome biogenesis regulators [33–35], including POLR I, nucleophosmin 1 (NPM1), fibrillarin (FBL), and nucleolin (NCL), during EMT. RT-qPCR showed that POLR I, NPM1, and FBL mRNA was increased significantly in TGFβ1 cells (Fig. 4J-L; Fig.S3J-L). NCL mRNA expression did not change obviously, but its protein expression was elevated in TGFβ1 cells (Fig. 4M, N; Fig.S3M, N). After LIX1L knockdown, only NCL mRNA was decreased in the three TGFβ1 cells (Fig. 4O-R, Fig.S3O-R). The above results indicated that the LIX1L expression plays a role in rRNA synthesis during EMT.
2.5. LIX1L interacts with NCL protein to regulate rRNA synthesis during EMT in NSCLC cells.
Similar to LIX1L, NCL is mainly located in nucleolus and regulates rRNA synthesis [33–35]. Next, we investigated the correlation between LIX1L and NCL expression in NSCLC cells. IF indicated that LIX1L and NCL protein were co-located in the nucleoli of TGFβ1 cells (Fig. 5A; Fig.S4A). Co-IP results confirmed the physical association between the endogenous LIX1L and NCL proteins (Fig. 5B; Fig.S4B) as well as between the exogenously expressed Flag-LIX1L and the endogenous NCL (Fig. 5C; Fig.S4C). Besides, LIX1L-NCL interaction was demonstrated in the nuclear extract of TGFβ1 cells (Fig. 5D; Fig.S4D). Next, we investigate the effect of LIX1L on NCL expression. Western blot showed that the NCL expression was obviously increased after LIX1L overexpression (Fig. 5E; Fig.S4E), but inhibited upon LIX1L knockdown (Fig. 5F; Fig.S4F). Besides, we performed LIX1L knockdown combined with MG132 (proteasome inhibitor) treatment and found that MG132 restored the reduced LIX1L and NCL protein levels due to LIX1L knockdown, suggesting that LIX1L and NCL protein might be degraded through proteasome pathway (Fig. 5G; Fig.S4G). NCL is critical for rRNA synthesis [36, 37]. We then knocked down NCL expression and observed a significant decrease in the 47s pre-rRNA level (Fig. 5H; Fig.S4H) and the size of nucleoli (Fig. 5I; Fig.S4I) in TGFβ1 cells. Furthermore, we investigated the role of NCL in LIX1L-induced rRNA synthesis during EMT. RT-qPCR showed that NCL knockdown partially reversed the enhanced rRNA synthesis due to LIX1L overexpression (Fig. 5J; Fig.S4J). These results indicated that LIX1L physically interacts with NCL and promotes its expression in EMT NSCLC cells, which might be the mechanism of LIX1L involved in the rRNA synthesis during EMT.
2.6. LIX1L-NCL promotes proliferation and core EMT functions of NSCLC cells.
To investigate the biological functions of NCL during EMT, we knocked down its expression in H358-TGFβ1, HCC827-TGFβ1, and H1975-TGFβ1 cells using specific siRNA transfection (Fig. 6A; Fig.S5A). NCL Knockdown resulted in a significant decrease in migration and invasion (Fig. 6B; Fig.S5B, P < 0.05), cell proliferation (Fig. 6C; Fig.S5C, P < 0.05), colony formation (Fig. 6D; Fig.S5D, P < 0.05), and anoikis resistance (Fig. 6E, F; Fig.S5E, F, P < 0.05), accompanied with upregulated E-cadherin expression and reduced Vimentin and Twist1 expressions (Fig. 6G; Fig.S5G). These results indicated that NCL plays important functions in the EMT process of NSCLC cells.
To elucidate whether NCL participates in LIX1L-mediated core EMT functions, we knocked down NCL expression in LIX1L overexpression cells. Transwells results showed that NCL knockdown reversed the increased migration and invasion abilities due to LIX1L overexpression (Fig. 6H; Fig.S5H, P < 0.05). CCK8 and clone formation assays showed that NCL knockdown abrogated LIX1L overexpression-induced proliferation (Fig. 6I, J; Fig.S5I, J, P < 0.05). The above results indicated that NCL plays a role in LIX1L-mediated EMT functions of NSCLC cells.
2.7. NCL protein is correlated with LIX1L expression, mesenchymal markers, lymphatic metastasis, and high TNM stage in NSCLC tumor specimens.
Next, we evaluate NCL expression in the 81 NSCLCs using IHC. NCL staining was mainly detected in the nucleus of tumor cells (Fig.S6A, B). Positive expression rate of NCL was 70.4% (57/81) and positively correlated with lymphatic metastasis and high TNM stage (Table S2, P < 0.0001). Besides, a positive correlation was observed between NCL and LIX1L (Fig.S6C, D; LUAD, r = 0.417, P = 0.024; LUSC, r = 0.526, P < 0.01), as well as between NCL and Vimentin (Fig.S6C, D; LUAD, r = 0.358, P = 0.012; LUSC, r = 0.437, P = 0.0338) in 41 LUAD and 40 LUSC specimens, respectively.
2.8. Inhibition of rRNA synthesis by CX-5461 inhibits cell proliferation and core EMT functions of NSCLC cells
To examine the effect of rRNA synthesis on EMT process of NSCLC cells, we employed CX-5461 to inhibit rRNA synthesis [26]. CX-5461 treatment in TGFβ1 cells led to a significant reduction in 47s pre-rRNA expression (Fig. 7A; Fig.S7A, P < 0.05), accompanied by decreased migration, invasion (Fig. 7B; Fig.S7B, P < 0.05), proliferation (Fig. 7C, D; Fig.S7C, D, P < 0.05), and anoikis resistance (Fig. 7E, F; Fig.S7E, F, P < 0.05). In addition, CX-5461 also significantly reduced the expression of Vimentin and Twist1 protein (Fig. 7G; Fig.S7G).
Next, we performed LIX1L overexpression combined with CX-5461 treatment in H358, HCC827, and H1975 cells to elucidate whether rRNA synthesis participated in LIX1L-mediated biological functions. Transwells assay demonstrated that CX-5461 significantly reversed the enhanced migration and invasion due to LIX1L overexpression (Fig. 7H; Fig.S7H, P < 0.05). CCK8 and clone formation assays showed that CX-5461 treatment partially abrogated the LIX1L overexpression-induced proliferation (Fig. 7I, J; Fig.S7I, J, P < 0.05). These results suggested that rRNA synthesis is required for LIX1L to execute EMT in NSCLC cells.
2.9. Upregulated LIX1L enhances EGFR-TKIs resistance in TGFβ1-treated EMT NSCLC cells in part by upregulating NCL expression and rRNA synthesis.
To investigate the effects of LIXIL on EGFR-TKIs resistance, cell response to gefitinib or erlotinib treatment was detected using CCK8 method. Compared to parental cells, the viabilities of H358-TGFβ1, HCC827-TGFβ1, and H1975-TGFβ1 cells were all significantly increased upon gefitinib (Fig. 8A; Fig.S8A, P < 0.05) or erlotinib (Fig. 8B; Fig.S8B, P < 0.05) treatment. IC50 value indicated that TGFβ1 cells were more resistant to gefitinib and erlotinib compared to parental cells (Fig. 8C; Fig.S8C, P < 0.05). LIX1L knockdown in TGFβ1 cells decreased the cell resistance to gefitinib and erlotinib treatment, and the IC50 value is decreased correspondingly (Fig. 8D-F; Fig.S8D-F, P < 0.05). In contrast, overexpression of LIX1L significantly promoted cell resistance to gefitinib and erlotinib in H358, HCC827, and H1975 cells (Fig. 8G-I; Fig.S8G-I, P < 0.05). These results indicated that LIX1L expression is positively correlated with EGFR-TKIs resistance in NSCLC cells.
Next, we explore whether NCL expression and rRNA synthesis participate in the LIX1L-mediated EGFR-TKIs resistance. As shown in Fig. 8J-M and Fig.S8J-M, overexpression of LIX1L significantly promoted cell resistance to gefitinib and erlotinib, which is consistent with the above results in Fig. 8G-I and Fig.S8G-I. However, silencing NCL (Fig. 8J, K; Fig.S8J, K, P < 0.05) or treating with CX-5461 (Fig. 8L, M; Fig.S8L, M, P < 0.05) significantly reversed LIX1L-induced cell resistance to gefitinib and erlotinib treatment. Together, the above data suggested that LIX1L expression enhances EGFR-TKIs resistance in TGFβ1-treated EMT NSCLC cells in part through upregulated NCL expression and rRNA synthesis.