Despite the morbidity and mortality of GC have declined over the past decade, we still face many problems and challenges in the screening and treatment of GC. TNM staging, encompassing the depth of invasion (T), lymph node metastasis (N), and distant metastasis (M) stages, is regarded as the most significant prognostic factor of GC. Because of tumor heterogeneity, even patients with the same pathologic stage of GC may have considerable differences in survival after complete surgical resection, indicating that prognosis cannot be accurately determined based on the current staging system alone. Even after complete resection or targeted therapy, many advanced GC patients still die of local recurrence and/or distant metastasis. Among which, tumor metastasis and chemotherapy failure are the mainly severe problem all clinical doctors have to face. Although various genes and pathways have been researched in GC, the more powerful mechanisms are still needed to resolve the metastasis and chemotherapy resistance.
Recently, LIM domain-only protein family plays pivotal roles in tumor progression, including radiotherapy resistance and metastasis [7, 28]. Some studies showed that FHLs play as a tumor repressor in breast cancer, liver cancer, and lung cancer [32–34], while other previous studies pointed out that FHLs promotes paclitaxel resistance and radiotherapy resistance in liver cancer cells and HeLa cells respectively [28, 35], promote tumor cell growth in liver cancer, glioma and breast cancer [36, 37], and promote metastasis in breast cancer cells and pancreatic cancer [7, 30]. However, the role of FHLs in gastric cancer is still unclear. In our study, we find the expression level of FHL3 is obviously up-regulated in GC both in mRNA and protein by analysis in TCGA, GTEx, and Beijing dataset (P < 0.05, Fig. 1a-h). Meantime, the same results are gained from 16 fresh-frozen tumor tissues and 120 paraffin-embedded sections (P < 0.01). Then, 120 samples show higher level of FHL3 refers to lower differentiation (P = 0.009, Table 1), metastasis trend (P = 0.002, Table 1) and worse stage of TNM (P = 0.039, Table 1) in GC. Besides, FHL3 is negatively associated with the prognosis in GC through Kaplan-Meier analysis in TCGA, KM-plotter cohorts and 120 GC samples (P < 0.05, Fig. 1i, j and 2c). Furthermore, the univariate analysis shows higher level of FHL3 accompanies with higher risk of GC progression (Table 2, HR = 2.06, p = 0.005). In other words, those results suggest that FHL3 is a potential predictor of disease progression and prognosis in GC.
In the following experiments, the tumor growth is decreased ~ 50% in HGC and ~ 20% in SGC by the FHL3 knockdown (Fig. 3g, i and j). In subcutaneous tumor model, our study finds that FHL3 knockdown reduces the tumor growth ~ 25% in tumor volume and ~ 25% in tumor weight (Fig. 5c and d). Besides, FHL3 knockdown enhances the cytotoxicity of OPH both in HGC and SGC (Fig. 3g, i and j), which is similar to the results in subcutaneous tumor model (Fig. 5a-d). In addition, our study finds the level of FHL3 is positive with the level of Slug, Snail, Twist1, N-cadherin and vimentin, while it is negative with the level of E-cadherin (Fig. 4a1 − 6). And FHL3 knockdown obviously decreases the expression level of vimentin, Snail and Slug, and increases the level of E-cadherin and ZO-1 both in HGC and SGC (Fig. 4b1 − 7). Meantime, migration and invasion ability are reduced by FHL3 knockdown. (Fig. 4c-e2).
However, the mechanisms of how FHL3 interferes in the regulation of tumor chemotherapy resistance and metastasis are unclear in GC. To our knowledge, EMT is mainly responsible for tumor metastasis, and the level of E-cadherin is considered as the feature in EMT process [6, 9]. E-box, a pivotal DNA reading frame in CDH1 sequence, through which some transcriptional factors can directly bind to CDH1 to regulate the expression level of E-cadherin, such as snail1/2, twist1/2, Zeb1 and FOXC2 [9]. Besides, other cellular junction protein also paly roles in tumor metastasis, such as N-cadherin and ZO-1. The regulation of EMT hold some recognized pathways, among which the TGFβ-mediated smad-dependent and smad-independent ways are major ones [9, 6]. For TGFβ/smad-dependent way, smad complex directly regulates the expression level of EMT-TFs to promote EMT process [9, 6]. Previous studies have showed that FHLs promote phosphorylation of smad2/3 and directly interact with them to lead nuclear accumulation of smad complex in liver cancer, which is important in EMT induction [32]. For TGFβ/smad-independent way, PI3K/Akt, MAPK, NF-κB, Hedgehog and Wnt/β-catenin pathways are upstream regulators of EMT-TFs [38–41]. Some studies point out that FHLs can increase the activation and transcription of Akt to promote tumor growth and progression in glioma, breast cancer, and ovarian cancer [42, 43, 30]. Other published papers show that FHLs interfere the MAPK/ERK pathway to result in radiotherapy resistance in pancreatic cancer [44]. It is reported that FHLs knockdown contributes to tumorigenesis prevention in osteosarcoma by down-regulation of Wnt/β-catenin pathways [27]. And FHLs are also associated with hepatocarcinogenesis by activating NF-κB pathway [45]. Therefore, FHL3 is likely to regulate gastric cancer metastasis through the TGFβ/smad-independent pathway. Encouragingly, our study finds MAPK, PI3K/Akt and TGFβ pathways is close to FHL3 by KEEG and GO analysis (Fig. 5a and b). And in the following experiments, FHL3 knockdown obviously down-regulates the phosphorylation level of MAPK downstream targets ERK1/2, P38 and JNK in HCG cells (Fig. 5c1-4). Besides, FHL3 knockdown obviously down-regulates the phosphorylation level of PI3K, Akt and GSK3β in HCG cells (Fig. 5d1 − 4). However, FHL3 knockdown induced down-regulation of TGFβ has few effects in the phosphorylation level of Smad4. Collectively, FHL3-induced EMT is associated with the activation of MAPK/ERK/JNK/P38 and PI3K/Akt/GSK3β pathways. Furthermore, based on our previous study that FHL3 regulates the Akt/GSK3β/ubiquitin-Snail1/Twist1 pathway to stabilize the EMT-TFs to promote EMT process in pancreatic cancer, we explore the role of FHL3 in the regulation of ubiquitin-mediated EMT. Interestingly, we find E3 ligase RNF146 can form a complex with FHL3 and Slug, and the up-regulation of FHL3 establishes the up-regulated RNF146-induced ubiquitination degradation of Slug (Fig. 8a and b).
Well, the mechanism of chemotherapy resistance is complicated. On the one hand, ABC transporters, especially for MDR1, is a central role in chemo-drug efflux to make chemotherapy resistance in gastric cancer [46, 47]. MDR1 is directly regulated by NF-κB pathway. And hypoxia also participates in the regulation of MDR1, through which up-regulation of HIF-α can lead chemotherapy in gastric cancer [23]. On the other hand, previous studies show that the inhibition of apoptosis is also important in chemotherapy, and those apoptosis-inhibition-mediated chemotherapy is regulated by MAPK pathway and PI3K/Akt pathway [20–22]. Besides, EMT is also considered to be responsible for chemotherapy resistance in pancreatic cancer and breast cancer [12, 13]. Nevertheless, the characteristic changes of some signaling pathways in mesenchymal phenotype may be related to the chemotherapy resistance shown by those EMT phenotype cells. Up-regulation of EMT-TFs, such as Snail and Twist1, simultaneously increase the level of ABC transporters to endow chemotherapy resistance during its up-regulation of EMT process [19, 17, 18]. In fact, our study finds that down-regulation of FHL3 promotes the mesenchymal-epithelial transition (MET), during which it may reduce chemotherapy resistance in HCG cells. Furthermore, our study finds MDR1 is down-regulated in FHL3-knockdown process (Fig. 8b, e). In other words, those TGFβ/smad-independent pathways are the working regulators in FHL3-mediated chemotherapy resistance.