1. LAMC1 expression was upregulated by TGFβ through synergistic activation of SMAD4 and SP1 and predicted a poor prognosis in ESCC.
Much of the previous research has established that TGFβ plays an important role in the tumor microenvironment, especially in cell-to-cell signaling25. We sought to identify genes regulated by TGFβ1, and those genes were found to be involved in signaling molecules and interaction pathways of the tumor microenvironment in ESCC. Furthermore, these genes themselves could influence the prognosis of patients with ESCC. We made these observations through the following process. First, by analysis of our mRNA microarray data (GSE53625), we found that 4130 genes were upregulated in cancer tissues compared with adjacent tissues (Log2FC > 0.5, FDR < 0.001), and 238 genes were significantly (p < 0.05) associated with poor prognosis (Fig S1A). Second, by using Pearson's correlation analysis of GSE53625 and TCGA data, we found that 1609 genes were positively correlated with TGFβ1 in ESCC cancer tissues (r > 0.15, FDR < 0.05) (Fig. 1A, S1B). At the same time, in our previous study, RNA-seq was performed on TGFβ1-treated and untreated ESCC cells26, and we found that a total of 3084 genes were upregulated (Log2FC > 0, FDR < 0.05) (Fig. 1B). Combining the two results, we found that a total of 625 genes were co-expressed with TGFβ1 and upregulated by TGFβ1 (Fig. 1C). Enrichment analysis of these genes (KOBAS)47 (FDR < 0.0001) revealed that a total of 11 pathways were involved in environmental information processing, only two pathways of which, cytokine-cytokine receptor interaction and ECM-receptor interaction, were included in signaling molecules and interaction pathways (Fig. 1D, Fig S1C). A total of 31 genes were enriched in these two pathways. The 31 genes are positively regulated by TGFβ1 and are involved in signaling and cellular interactions in the TME. Overlap of the genes associated with prognosis showed that LAMC1 was the only one of the 31 genes that affected the prognosis of ESCC patients (Fig. 1E). Accordingly, at the protein level, LAMC1 was more highly expressed in cancer tissues than in para-cancer tissues, as demonstrated by IHC staining (Fig. 1F) and was also associated with low OS (Fig. 1G) and tumor stage (Supplementary Table 1). Therefore, LAMC1 can be used as an independent prognostic marker for ESCC.
Compared with that in the control group, the expression of LAMC1 was increased at the protein and mRNA levels in KYSE30 and KYSE450 cells after TGFβ1 treatment, which was time- and concentration-dependent in ESCC cells (Fig. 1H, Fig S2A, B). Additionally, to determine whether the TGFβ signaling pathway is responsible for the expression of LAMC1, we used the TGFβ receptor inhibitor SB505124 to eliminate the effect of TGFβ1 on LAMC1. The results showed that SB505124 could reverse TGFβ1-induced LAMC1 expression in KYSE30 and KYSE450 cells (Fig. 1I, J). These results suggest that TGFβ signaling is responsible for the induction of LAMC1 transcription. By TRANFAC and JASPER database prediction, we speculated that the transcription factors SMAD4 and SP1 synergistically induced LAMC1 transcription. A ChIP assay was performed using anti-SMAD4 and anti-SP1 antibodies, and we found that TGFβ1 led to a significant increase in the enriched LAMC1 promoter sequence, suggesting that SMAD4 and SP1 were recruited to the promoter of the LAMC1 gene by TGFβ1 treatment (Fig. 1K). Additionally, by measuring proteins of chromatin fractions with antibodies against SP1 and SMAD4, we found that the expression of SMAD4 and SP1 were increased in each other’s chromatin fraction (Fig. 1L). Furthermore, we conducted knockdown SP1 or SMAD4 ESCC cells respectively, and conducted ESCC cells that combined knockdown SP1 and SMAD4 (Fig. 1M, N, Fig S3 A, B). The expression of LAMC1 was decreased in those cells, which could not be rescued by TGFβ1 treatment (Fig. 1O-Q). This result suggested that the transcription factors SP1 and SMAD4 together induced the transcription of LAMC1. Taken together, these results showed that LAMC1 would be directly regulated by the TGFβ/SMAD4-SP1 signaling pathway.
2. LAMC1 promoted the proliferation and migration of ESCC cells in vitro and in vivo.
To evaluate the tumorigenic effect of LAMC1 on ESCC, we constructed KYSE30 and KYSE450 cell lines with stable knockdown or overexpression of LAMC1 (Fig. 2A, B). shLAMC1 in KYSE30 and KYSE450 cells inhibited cell proliferation. Accordingly, overexpression of LAMC1 promoted cell proliferation (Fig. 2C,). And shLAMC1 in KYSE30 and KYSE450 cells promoted apoptosis (Fig. 2D). Furthermore, we also found that overexpression of LAMC1 could promote ESCC cell migration, while shLAMC1 inhibited migration (Fig. 2E).
In vivo, we established a xenograft tumor mouse model by subcutaneous inoculation or intravenous tail injection of KYSE30 cells transfected with shLAMC1, sh-vec, overexpression-LAMC1 and control vector. Consistent with the results of the in vitro experiments, the tumor volume and weight in the overexpressing LAMC1 group were significantly increased compared with those in the control group. The shLAMC1 group exhibited the opposite pattern (Fig. 2G-J). The number of pulmonary metastasis nodules in the groups showed similar results (Fig. 2K, L).
3. The positive effect of LAMC1 on the migration in ESCC cells mainly via the Akt/IKKα/NF-κB/MMP9-MMP14 pathway.
We performed mRNA sequencing in KYSE30 and KYSE450 cells with sh-1 LAMC1 or sh-vec to explore downstream signaling pathways responsible for the aggressiveness of ESCC. Gene set enrichment analysis (GSEA) suggested that LAMC1 knockdown could affect the apoptosis pathway, NF-κB pathway, and cytokine and chemokine pathways (Fig. 3A). We detected IKKα phosphorylation of Akt, IKKα and p65 levels in shLAMC1 and LAMC1-overexpressing ESCC cells and found that expression of LAMC1 was positive correlated with phosphorylation of Akt, IKKα and p65 (Fig. 3B, C). Matrix metalloproteinases (MMPs) play an important role in tumor cell invasion and metastasis and are common downstream regulators of NF-κB-mediated cell metastasis17. We detected MMP2, MMP9, MMP10, MMP13, and MMP14. The results showed that the expression of MMP9 and MMP14 was in accordance with the phosphorylation of Akt, IKKα and p65 in shLAMC1-expressing and LAMC1-overexpressing ESCC cells (Fig. 3B, C). Furthermore, through cell immunofluorescence, we also found the expression of NF-κB (p65) were decreased in the nucleus in knockdown LAMC1 cells than the controls (Fig. 3D). Additionally, TNFα, as an activator of the NF-κ B pathway, could restore the expression of phosphorylation of IKKα, NF-κB, MMP9 and MMP14 in knockdown LAMC1 (Fig. 3E). Accordingly, the Akt phosphorylation selective inhibitor MK-2206 and the NF-κB nuclear translocation inhibitor JSH-23 both could reversed the high expression of phosphorylation of Akt, IKKα, NF-κB, MMP9 and MMP14 in overexpression LAMC1 cells (Fig. 3F).TNFα, also, reversed the inhibitory migration of shLAMC1 ESCC cells (Fig. 3G, S4 A). Accordingly, JSH-23abrogated the promotion of migration in LAMC1-overexpressing ESCC cells (Fig. 3H, S4 B I).
4. LAMC1 inhibited apoptosis mainly through the Akt/NF-κB/caspase9-caspase3-PARP cascade.
Cleaved caspase-9, cleaved caspase-3, and cleaved PARP levels increased in shLAMC1 KYSE30 and KYSE450 cells compared with the control group after cisplatin treatment (Fig. 4A), while they decreased in LAMC1-overexpressing cells compared with vector only cells (Fig. 4B). Furthermore, previous studies have shown that the NF-κB pathway can regulate cell anti-apoptosis via caspase19. We found that cleaved caspase9, cleaved caspase-3, and cleaved PARP expression in shLAMC1 cells was decreased by TNFα stimulation (Fig. 4C). Accordingly, after treatment with Akt phosphorylation selective inhibitor MK-2206 and the NF-κB nuclear translocation inhibitor JSH-23, LAMC1-overexpressing ESCC cells showed the opposite results in proliferation and expression of cleaved caspase and PARP (Fig. 4D). Furthermore, JSH-23 also restored the positive effect on proliferation of overexpression LAMC1 cells (Fig. 4E), and TNFα could restore the negative effect on proliferation and the positive effect on apoptosis of shLAMC1 KYSE30 and KYSE450 cells (Fig. 4F, G).
5. CXCL1 would be regulated by LAMC1 mainly via NF-κB activation.
Increasing attention has been paid to the role of the TME in solid tumors. Cytokines and chemokines, as tumor-promoting factors, often play a role in intercellular signaling. By GSEA enrichment analysis of mRNA-seq data of shLAMC1 and sh-vec ESCC cells, knockdown of LAMC1 affected the cytokine and chemokine signaling pathways (Fig. 3A). Moreover, enrichment analysis of RNA-seq data of ESCC tissues (GSE53625) revealed the same effect of LAMC1 (Fig. 5A). We hypothesized that LAMC1 is involved in the regulation of signaling molecules and interactions in the TME via enrichment analysis of genes positively regulated by TGFβ1. All the above findings suggest that LAMC1 may be involved in regulating the secretion of cytokines or chemokines. First, we detected a total of 48 cytokines and chemokines in the conditioned medium (CM) of shLAMC1 cells using the Bio-Plex Pro Human Chemokine Panel 48-plex kit. We found that the expression of CXCL1, IL8, and MIF increased in sh-vec cells compared with sh-1 cells at a higher concentration (Fig. 5B). We also detected the expression of the three cytokines in concentrated CM and found that only CXCL1 was regulated by LAMC1 both in KYSE30 and KYSE450 cells (Fig. 5C). Considering those results, we speculate that LAMC1 may upregulate CXCL1, and it was confirmed in knockdown LAMC1 and overexpression LAMC1 cells by ELISA (Fig. 5D). Thus, CXCL1 could be identified as a downstream target of LAMC1.
As predicted on the Cistrome website53, NF-κB can upregulate CXCL1 through transcriptional activation (Fig. 5E). In addition, we determined that LAMC1 may activate the NF-κB pathway, so we speculated that LAMC1 upregulates CXCL1 through NF-κB transcriptional activation. To verify whether NF-κB is responsible for CXCL1 expression, we used ELISA and WB to detect CXCL1 secretion of knockdown LAMC1 cells with or without TNFα stimulation and that of overexpression LAMC1 cells with or without MK-2206 2HCI and JSH-23. As expected, lower CXCL1 secretion by LAMC1 knockdown cells could also be increased by TNFα, and MK-2206 2HCI and JSH-23 could reverse the high expression of overexpression LAMC1 cells (Fig. 5F, G) And at the mRNA levels, expression of CXCL1 in shLAMC1 cells also could be revered by TNFα (Fig. 5H).
In vivo, the expression of LAMC1 was associated with that of CXCL1 at the RNA level (Fig. 5I), and the expression of CXCL1 was higher in cancer tissues than in adjacent tissues (Fig. 5J), but it did not affect the prognosis of patients, especially OS (Fig S4E).
6. CXCL1 secreted by ESCC tumor cells promoted the transformation of CAFs into inflammatory CAFs.
CAFs are heterogeneous cells with different subtypes, such as iCAFs and myCAFs33. The two subgroups not only have significant differences in their transcriptional profiles but also have different effects on tumor cells. myCAFs are contractile and can remodel the stroma, while iCAFs are characterized by a secretory phenotype and regulate tumor cells and other cells in a paracrine manner. CAFs were isolated from fresh tumor tissue and cultured in vitro and were identified by αSMA expression by cell immunofluorescence detection (Fig S5A). To explore whether ESCC tumor cell secreted CXCL1, upregulated by LAMC1, influences CAF heterogeneity, we performed mRNA-seq in the following cells: CAFs treated with PBS, CAFs with recombinant CXCL1 (rCXCL1) treatment and CAFs with sh-vec CM treatment. We found that in CAFs treated with rCXCL1 or sh-vec CM, some gene clusters of iCAFs, including cytokines (CSF2, VEGF, etc.), chemokines (CXCL2, CXCL3, CXCL5, etc.) and interleukins (IL6, IL7, etc.), were upregulated than CAFs with PBS stimuli. In addition, gene clusters of myCAFs, such as COL1A1 and COL4A1, were downregulated (Fig. 6A). Moreover, compared with analysis of the controls, GSEA of CAFs with rCXCL1 or sh-vec CM treatment confirmed the upregulation of the cytokine/chemokine signaling and the regulation of STAT cascade, especially phosphorylation of STAT3 pathway. But the smooth muscle contraction pathway was downregulated (Fig. 6B, C).
In order to verify the above sequencing results, we detected iCAF markers (IL1, IL6, LIF, CSF3) and myCAF markers (Acta2, Ctgf) in CAFs treated with CM from shLAMC1 ESCC cells, CAFs cocultured with shLAMC1 ESCC cells, and controls. Since inflammatory markers are secreted, the expression of these proteins was detected in the concentrated CM of CAFs. At the protein and mRNA level, we found that CAFs cocultured with sh-vec ESCC cells or treated with the CM of sh-vec ESCC cells had higher expression of inflammatory markers than CAFs cocultured with shLAMC1 ESCC cells (Fig. 6D, E) or treated with CM from shLAMC1-1 ESCC cells (Fig. 6F, G). But the myCAF markers (Acta2, Ctgf) had lower expression in CAFs cocultured with sh-1 ESCC cells or treated with the CM of sh-1 ESCC cells than controls at protein level, which were not obvious at mRNA level (Fig. 6G). To verify whether CXCL1 is the main driver of this effect, we directly treated CAFs with rCXCL1 and obtained a similar result (Fig. 6H, I).
7. Tumor-secreted CXCL1 induced iCAF formation via phosphorylation of STAT3.
The CXCL1 common receptor is CXCR2, and combined with the above results of GSEA, we suggest that the CXCR2/pSTAT3 pathway may be responsible for this effect. We found that CXCR2 and pSTAT3 expression in CAFs was upregulated at the RNA and protein levels after rCXCL1 treatment (Fig. 7A, S5B). Additionally, SB225002, an inhibitor of CXCR2, reversed the upregulation of the inflammatory markers, CXCR2 and pSTAT3 and the downregulation of αSMA by rCXCL1 in CAFs (Fig. 7D-F). In addition, SB225002 decreased the changes in these proteins, especially inflammatory markers, in CAFs cocultured with shLAMC1 ESCC cells and CAFs treated with CM from shLAMC1 ESCC cells (Fig. 7B-C, G-L). All the above results suggest that CXCL1, regulated by LAMC1 and secreted by tumor cells, promotes the transition of CAFs into iCAFs via CXCR2/pSTAT3.
8. iCAFs, induced by CXCL1, promoted ESCC progression in vivo and in vitro.
Furthermore, to explore the effect of iCAFs induced by CXCL1 on the proliferation of tumor cells, we compared the proliferation of tumor cells after coculture with CAFs stimulated by PBS or rCXCL1 together with or without SB225002, and we found that CAFs with rCXCL1 promoted WT ESCC proliferation, which could be reversed by SB225002 (Fig. 8A). Additionally, to explore the influence of iCAFs on migration, WT ESCC cells with different treatments were divided into four groups: the control group (WT ESCC cells treated with PBS) and WT ESCC cells treated with CM secreted by CAFs that were pretreated with rCXCL1 or PBS (CM-CAFs-PBS, CM-CAFs-pretreatCXCL1) in the presence or absence of SB225002. We found that both CM-CAFs-pretreatCXCL1 and CM-CAFs-PBS could promote the proliferation and migration of ESCC cells, and CM-CAF-pretreatCXCL1 had a stronger promoting effect than CM-CAFs-PBS, while SB225002 could also reverse the effect (Fig. 8B, C). In vivo, the tumor volume and weight of WT KYSE30 cells mixed with rCXCL1-pretreated CAFs were larger than those of cells mixed with CAFs without rCXCL1 pretreatment. SB225002 also weakened this effect (Fig. 8D, E). The migration markers MMP9 were measured by IHC and western blot in xenograft tumor tissue. Tumor with rCXCL1 pre-treated CAFs had higher expression of MMP9 than that of tumor with PBS pre-treated CAFs, which could be decreased by SB225002 (Fig. 8F, G).