TMPRSS4 regulates levels of integrin |[alpha]|5 in NSCLC through miR-205 activity to promote metastasis

Lung cancer is a critical problem in public health. It represents the most frequent tumour type in men and the second in women, and the 5-year survival rate remains inferior to 20% (Jemal et al, 2010). More than 60% of NSCLC patients present locally advanced, unresectable or metastatic (stage III/IV) tumours at the time of diagnosis, which fatally concludes in death within few months after diagnosis. In spite of the advent of targeted therapies, which has been a breakthrough for NSCLC management, only a small percentage of patients will benefit from them. Therefore, there is a need for identifying new potential therapeutic targets against which more effective treatments may be developed. Altered expression of proteases is currently considered as a hallmark of cancer, as malignant cells need proteolytic activities to promote growth, motility and invasion (Roy et al, 2009). Type II transmembrane serine proteases (TTSPs) are characterised by the presence of an N-terminal transmembrane domain that anchors these proteases to the plasma membrane. TTSPs include 18 members that can be divided into four groups. Examples of TTSPs related to cancer are hepsin, matriptase, HAT/DESC and TMPRSS4 (Szabo and Bugge, 2008). TMPRSS4 comprises a short N-terminal cytoplasmic domain, a transmembrane domain and a large extracellular domain that contains the catalytic activity. TMPRSS4 is synthesised as an inactive zymogen that needs to be activated (Netzel-Arnett et al, 2003). However, both the substrates and activators of this serine protease are largely unknown. TMPRSS4 participates in the regulation of cellular signalling events at the plasma membrane and in the extracellular matrix (Hooper et al, 2001). TMPRSS4 is upregulated in pancreatic, colon, lung, ovary and thyroid cancers, where it has been suggested as a diagnostic marker (Kebebew et al, 2005). In a previous work, we demonstrated that TMPRSS4 is highly expressed in lung tumours compared with normal lung, particularly in squamous cell carcinomas (SCC) as compared with adenocarcinomas (AC). We also demonstrated that high TMPRSS4 mRNA levels in SCC are associated with poor prognosis (Larzabal et al, 2011). Moreover, a knockdown strategy to reduce TMPRSS4 levels in lung tumour cells resulted in a significant impairment of metastasis in animal models (Larzabal et al, 2011). Studies of biological activity in colon cancer have reported that elevated TMPRSS4 expression induces epithelial-to-mesenchymal transition (EMT) of cancer cells and promotes metastasis (Jung et al, 2008; Kim et al, 2010). Further analysis of TMPRSS4-mediated signalling in colon cancer cells suggested that multiple downstream signalling pathways are activated. These included Src, ERK1/2, AKT, FAK and Rac1, resulting in E-cadherin downregulation and induced expression of integrin α5β1, a critical adhesion molecule involved in the acquisition of an EMT phenotype and cell motility (Maschler et al, 2005). Inhibition of PI3K or Src with specific compounds decreases cell invasiveness and actin rearrangement mediated by TMPRSS4. Moreover, functional blockade of integrin α5β1 demonstrated that this integrin has an important role in TMPRSS4-mediated effects (Kim et al, 2010). In spite of these data, the molecular regulation of TMPRSS4 in cancer cells is poorly understood. The aim of this study was to identify molecular mechanisms involved in the protumorigenic regulation of TMPRSS4 in NSCLC. Through a microarray analysis, we discovered that the miR-205 gene (MIR205HG) was consistently overexpressed upon TMPRSS4 downregulation. We demonstrate here that overexpression of miR-205 promotes an epithelial phenotype and inhibits tumour cell migration and metastasis formation in lung cancer models. Moreover, we have identified integrin α5β1 (a proinvasive protein) as a new miR-205 direct target in NSCLC; we also show a novel molecular mechanism that connects TMPRSS4 with integrin α5β1 through miR-205. H358 and H441 cell clones with a reduction of TMPRSS4 (shTMP4) and their corresponding controls (carrying the empty vector; shCtrl) were previously described by our group (Larzabal et al, 2011). Immortalised normal human bronchial epithelial cells (HBECs) have been previously characterised (Ramirez et al, 2004). These cell lines were kindly provided by Dr J.D. Minna (University of Texas Southwestern Medical Center, Dallas, TX, USA). Cells were maintained in keratinocyte serum-free medium supplemented with human recombinant epidermal growth factor (EGF) and bovine pituitary extract (Life Technologies, Carlsbad, CA, USA). To detect mature miR-205 expression, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA. For the quantification of this miRNA, stem-loop RT of 20 ng total RNA was run using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) with specific primers for miR-205 and RNU48 as endogenous control (Applied Biosystems). Subsequently, the qPCR amplifications were performed with TaqMan 2 × Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems) and specific primers for miR-205 and RNU48 (Applied Biosystems). Relative miR-205 levels were normalised to RNU48 expression. Data are given as 2−ΔΔCt or 2−ΔCt. To evaluate the clonogenic potential of miR-205 expressing clones, 500 cells per well were plated into six-well plates in adherent conditions. After 10 days in culture, colonies were fixed with 4% buffered formalin (Panreac, New Jersey, NY, USA) and stained with 2% crystal violet. The number of colonies per well was determined. H441 cells did not migrate through the transwell in Boyden chambers (unpublished observations). For this reason, we examined migration of this cell line with an in vitro model of wound healing. Cells were grown until confluence, and a 20-p micropipette tip was used to create a linear scratch in the monolayer. Pictures of the wounds were taken right after the scratching and 24 h later with a Nikon Eclipse photomicroscope (Nikon, Kingston, UK) using the ACT-2U1.6 software (Nikon). The empty surface between the wound edges was measured with the TScratch analysis software (Zurich, Switzerland). To study the effect of miR-205 on primary tumour growth, 10 × 106 H2170 cells and their corresponding miR-205-overexpressing clones (miR-205#2 or miR-205#3) were injected subcutaneously into the flanks of the mice in a 1 : 1 PBS/Matrigel solution (BD). A similar experiment was conducted to assess whether knockdown of ITGα5 (shITGα5 H2170 cells) would decrease primary tumour growth with respect to controls. Tumour volumes were calculated with the formula: V=length × (width)2/2. ITGα5 3′UTR-F: 5′-CTAGAGAGGAGCTGGGGATCCCTCCGCCCTGTGAAGGAACCATGCACTGTGAAGGACCCTTGTGC-3′ ITGα5 3′UTR-R: 5′-GGCCGCACAAGGGTCCTTCACAGTGCATGGGGCGGAGGGATCCCCAGCTCCTCT-3′ ITGα5-3′UTR-Mut-F: 5′-CTAGAGAGGAGCTGGGGATCCTCAACCCCCAGACACTTGAGGTAGCCCTTGTGC-3′ ITGα5-3′UTR-Mut-R: 5′-GGCCGCACAAGGGCTACCTCAAGTGTCTGGGGGTTGAGGATCCCCAGCTCCTCT-3′ These oligonucleotides contain the target site sequence or the mutated version (indicated in bold letters), which is complementary to the seed sequence of miR-205. Hybridised oligonucleotides were ligated into the XbaI—NotI site of the renilla reporter vector (pRL-SV40; Promega). GeneJuice reagent was used to co-transfect the renilla- ITGα5-3′UTR vector or the Renilla- ITGα5-3′UTR-mut, and a control vector containing firefly luciferase (pGL3-basic; Promega), together with either pre-miR-205 or scramble precursor-miR vectors. Renilla luciferase activity was measured 48 h after transfection with the Dual-Luciferase System (Promega) in a Berthold Luminometer (Lumat LB 9507). A list of 287 probes was found to be deregulated by >two-fold in the three cell clones analysed. From those, 176 probes corresponded to non-annotated sequences. We then focused our analysis in those genes whose expression trend was the same (either upregulation or downregulation) in the three clones (Figure 1A). Twenty-nine genes followed this pattern (12 of them overexpressed and 17 of them underexpressed). As expected, TMPRSS4 was one of the downregulated genes of this set. Other downregulated genes that have a role in cancer cell adhesion included DDR1 and CLEC18A. DDR1 has been shown to promote lung cancer metastasis to the bone, and its expression correlates with poor prognosis in NSCLC patients (Valencia et al, 2012). One of the upregulated genes found in the list was MIR205HG. This microRNA has been described as a tumour suppressor gene in different tumour types (Song and Bu, 2009; Wu et al, 2009; Majid et al, 2011), a reason whereby we decided to further study its role in lung cancer. miR-205 expression in H358 and H441 shCtrl and shTMP4 cell clones was validated by qPCR (Supplementary Figure 1B). Initially, we assessed whether ectopic expression of miR-205 had a biological effect on proliferation and clonogenicity. MTT proliferation assays indicated that cell clones overexpressing miR-205 had a reduction in cell proliferation rates compared with cells transfected with scramble vector (miR-Scr) in both cell lines tested (Figure 1C). Furthermore, the overexpression of miR-205 decreased clonogenic capacity of H2170 and H441 cell lines (Figure 1D). These data indicate that miR-205 inhibits cell growth in lung cancer cell lines. As anchorage-independent growth is strongly correlated with tumorigenicity, we then determined whether miR-205 would alter cell growth in soft agar. H2170 cell clones were plated in soft agar and incubated for 2 weeks before counting the number of colonies. As shown in Figure 2A, the ability to form colonies was almost abolished in miR-205-overexpressing clones compared with scramble control. The same results were observed in the H441 cell line (Supplementary Figure 1E). A cell cycle analysis by flow cytometry was performed to determine whether the decrease in cell proliferation due to miR-205 upregulation was associated with a cycle arrest. Overexpression of miR-205 produced a significant increase in the percentage of cells in the G0/G1 phase (from 56.44±1.72% to 66.03±1.77%; P<0.05), along with a decrease in the S phase (Figure 2B), indicating that miR-205 induced a cell cycle arrest in the H2170 cell line. Similar results were observed in the H441 cell line (Supplementary Figure 1F). To study the effects of miR-205 on cell–matrix adhesion, we cultured H2170 cells on different substrates. miR-205 overexpression decreased cell adhesion to fibronectin (Figure 2E) but not to collagen type I (Supplementary Figure 2B). A similar tendency was found for H441 cells, although results did not reach statistical significance (data not show). Different studies have demonstrated that miR-205 regulates EMT by targeting ZEB2 (Gandellini et al, 2009; Matsushima et al, 2011). As shown in Figure 3A, ZEB1 and ZEB2 mRNA levels were strongly reduced in miR-205-overexpressing cells. The analysis of EMT markers by qPCR and immunofluorescence revealed a marked increase in E-cadherin along with a pronounced decrease in fibronectin in H2170 cells with miR-205 overexpression (Figures 3B–D). The effect of miR-205 on E-cadherin and fibronectin expression was similar (although less remarkable) in H441 cells compared with H2170 cells (Supplementary Figures 2C and D). These results show that miR-205 inhibits EMT and cell motility and promotes an epithelial phenotype. Furthermore, we used a xenograft model to analyse the effect of miR-205 on lung metastasis. In this case, animals were injected with the H2170 clones in the tail vein and monitored by bioluminescence until day 21. At day14th and 21st, a significant increase in light emission in the H2170 miR-Scr group was observed in comparison with the group injected with miR-205-overexpressing cells (Figures 4A and B). At the end of the study (day 21), animals were killed and lungs were examined for metastatic lesions. Histological analysis confirmed a less tumour burden in mice injected with cells that overexpressed miR-205 (Figure 4C). Immunohistochemical analysis of active caspase-3 revealed a higher proportion of apoptotic cells in tumours with increased levels of miR-205 (Figure 4D). Because ITGα5 is overexpressed in lung cancer (Dingemans et al, 2010), it promotes invasion of cancer cells (Hood and Cheresh, 2002) and its expression has been linked to TMPRSS4 in colon cancer (Kim et al, 2010), we focused our attention on this gene. We analysed whether endogenous ITGα5 mRNA and protein levels decreased when miR-205 was overexpressed. We found that miR-205 upregulation in H2170 or H441 lung cancer cells caused a significant reduction in endogenous ITGα5 mRNA levels (Figure 5B for H2170 cells and Supplementary Figure 2F for H441 cells) and protein levels (Figure 5C for H2170 cells and Supplementary Figure 2G for H441 cells). Moreover, Src signalling pathway activated by integrins was downregulated in H2170 cells with an overexpression of miR-205 (Figure 5D). To determine whether ITGα5 was a direct target of miR-205, we constructed renilla-based reporters that contained the wild-type or the mutated miR-205 target sequences of the ITGα5 3′-UTR. Ectopic expression of miR-205 inhibited the expression of the reporter vector containing the wild-type sequence of ITGα5 3′-UTR but not the reporter vector containing the mutation of the seed-miR-205 binding site in the two cell lines tested (Figure 5E for H2170 cells and Supplementary Figure 2H for H441 cells). These data show that ITGα5 is a direct target of miR-205 and that miR-205 can reduce ITGα5 mRNA and protein levels. As integrins are major adhesion molecules involved in cell-ECM interactions, we studied the effects of ITGα5 on cell–matrix adhesion. After culture of these cells on different substrates, we observed that ITGα5 levels modified the adhesiveness to fibronectin (Figure 6C) but not to collagen type I (data not shown). All these results provide evidence that inhibition of ITGα5 by miR-205 is responsible, at least in part, for the antitumorigenic effect observed in NSCLC cells with miR-205 upregulation. TMPRSS4 has been shown to increase cell motility and invasion, to induce EMT and to promote metastasis in different cancer models, through multiple downstream signalling pathways involving ERK, AKT, Src and Rac1 (Kim et al, 2010). TMPRSS4 is upregulated in some cancer types, such as colon, pancreas, thyroid and lung (Wallrapp et al, 2000; Kebebew et al, 2005; Kim et al, 2010; Larzabal et al, 2011). Thus, this serine protease has emerged as a promising diagnostic and therapeutic target in cancer. Nonetheless, the molecular regulation of TMPRSS4 leading to these effects is still unknown. In the present study, we have identified a new molecular mechanism whereby TMPRSS4 regulates integrin α5 levels through the expression of miR-205. Because ITGα5 is strongly involved in the protumorigenic effects elicited by TMPRSS4, the data reported here about this novel molecular axis will help designing therapeutic strategies to inhibit this pathway. Moreover, in the present study we confirm the tumour suppressor role of miR-205 in lung cancer. In order to elucidate the mechanism of action elicited by TMPRSS4 during EMT and metastasis formation, we conducted a microarray analysis where we compared gene expression profiles after TMPRSS4 inhibition in H358 and H441 lung cancer cell lines, with those of mock-transfected cells. As expected, the majority of genes with a differential expression were related with cell invasion and motility, in keeping with the functional role described for this serine protease in some tumour types (Jung et al, 2008; Kim et al, 2010). One of the upregulated genes was miR-205, a microRNA that has been described as a tumour suppressor (Song and Bu, 2009; Wu et al, 2009; Majid et al, 2011). MicroRNAs (miRs) are small, non-coding RNAs that negatively regulate gene expression via translational repression or messenger RNA degradation (Bartel, 2004). MicroRNAs are involved in biologic and pathologic processes, including cell differentiation, proliferation and apoptosis (Kloosterman and Plasterk, 2006). Accumulating evidence indicates that deregulation of miRs is associated with the development of human cancers and suggests a causal role of miRs in tumour initiation and progression, as they can function as oncogenes or tumour suppressors (Caldas and Brenton, 2005; Chen, 2005). Recently, studies have shown that miR-205 regulates EMT through the modification of E-cadherin and other adhesion proteins (Bracken et al, 2009; Gibbons et al, 2009). Expression of the miR-200 family and miR-205 has been shown to be reduced in cells undergoing EMT. These miRs act by directly targeting the 3′-UTR sequence of ZEB1 and SIP1 (Gregory et al, 2008; Park et al, 2008). miR-205 has also been shown to suppress metastatic spread of human breast cancer xenografts in nude mice (Iorio et al, 2009) and to exert a tumour suppressor role by targeting HER3 receptor and VEGF-A in breast cancer (Iorio et al, 2009; Wu and Mo, 2009) and Src in renal cancer (Majid et al, 2011). In lung cancer, overexpression of miR-205 in cell lines hinders cell migration and invasion (Song and Bu, 2009) and its inhibition results in the acquisition of cancer stem cell and EMT properties, which favors tumour progression (Tellez et al, 2011). Consistent with these reports, we demonstrate that miR-205 was markedly downregulated in lung cancer cell lines compared with non-malignant HBEC lung epithelial cells. Its overexpression strongly reduced cell proliferation and clonogenic survival and caused G0/G1 cell cycle arrest. In addition, we show that forced expression of miR-205 in lung cancer cells impaired migratory and invasive capabilities. Furthermore, we demonstrate that miR-205 expression reduced lung metastasis formation and promoted an epithelial phenotype by inducing E-cadherin and decreasing fibronectin levels. However, the role of miR-205 in lung cancer remains controversial, since some studies indicate a high expression of this miRNA in lung cancer compared with normal lung tissue (Yanaihara et al, 2006; Markou et al, 2008; Lebanony et al, 2009). Interestingly, expression of miR-205 has been described as a biomarker to distinguish between AC and SCC (Hamamoto et al, 2013; Jiang et al, 2013). It is worth noticing though, that exposure of HBEC cells to tobacco carcinogens silences miR-205 expression through epigenetic mechanisms, leading to a dedifferentiation programme (Tellez et al, 2011). Moreover, epigenetic silencing of miRNAs with tumour suppressor features, including miR-205, is emerging as a common hallmark of human tumours (Lujambio et al, 2008). Each miRNA has the potential to target hundreds of genes, which harbour sequences in their 3′-UTRs that are complementary to the seed region of the miRNA (Lim et al, 2005). Different targets of miR-205 have been described including, as previously mentioned, ZEB1 and SIP1 (Gregory et al, 2008), ERBB3 and VEGF-A (Wu et al, 2009), LDL receptor protein 1 (Song and Bu, 2009) and PKCε (Gandellini et al, 2009). In the present study, we have identified and validated for the first time ITGα5 as a new target of miR-205 in cancer. Integrins are a family of cell adhesion proteins that activate diverse intracellular signalling molecules and reorganise the actin cytoskeleton to regulate attachment, survival and motility (Giancotti and Ruoslahti, 1999; Hood and Cheresh, 2002). In NSCLC, expression of particular integrins has been shown to predict the clinical course and prognosis of patients (Adachi et al, 2000; Gogali et al, 2004). ITGα5 binds to integrin β1 to give rise to the α5β1 heterodimer. Fibronectin stimulates the proliferation of lung cancer cells through α5β1 integrin receptor-mediated signalling; thus knockdown of this integrin reduces tumour burden (Roman et al, 2010). High expression of ITGα5 is associated with lower overall survival in patients with early stages of NSCLC (Dingemans et al, 2010). Moreover, high ITGα5 levels have been associated with poor prognosis in NSCLC patients with negative lymph nodes (Adachi et al, 2000). We show here that 22 out of 30 lung cancer cell lines analysed expressed ITGα5. Furthermore, inhibition of this integrin diminishes cancer cell migration, adhesion and tumour growth. These data suggest that ITGα5 may constitute a target worth studying in patients with NSCLC. A close relationship between acquisition of EMT features and ITGα5 expression has been observed. Inhibition of E-cadherin in ovarian cancer cells causes upregulation of ITGα5 (Sawada et al, 2008). ZEB2, a transcription factor that represses E-cadherin, upregulates ITGα5 levels through cooperation with Sp1 to induce EMT and invasion in cancer cells (Nam et al, 2012). In this line, Kim et al (2010) pointed to TMPRSS4 as a new regulator of EMT in colon cancer and suggested that Src, FAK and ERK, which are major downstream effectors of integrins, appeared to be key signalling molecules involved in cell invasion and in the cadherin switch, presumably via regulation of ITGα5 expression. In agreement with these results, we show a downregulation of ITGα5 in H358, H441 and H2170 lung cancer cell lines with an inhibition of TMPRSS4, which confirms the regulation of ITGα5 by TMPRSS4. Therefore, it appears that targeting the novel TMPRSS4/miR-205/ITGα5 axis may be a promising strategy to inhibit EMT and metastasis in NSCLC. In this regard, an antibody targeting integrin α5β1 (volociximab, PDL Biopharma) has been developed and is being currently tested in phase II clinical trials for solid tumours (particularly for renal carcinoma). In summary, our results provide evidence for the existence of a new molecular connection between two membrane-anchored proteins (ITGα5 and TMPRSS4) that cooperate to foster tumour growth, metastasis and migration, through miR-205. This new intracellular signalling pathway appears to have an important role in the development of lung cancer. TMPRSS4 blockade in tumour cells causes an overexpression of miR-205, resulting in an inhibition of the transcription factors ZEB1 and ZEB2, and ITGα5, which leads to a loss of EMT features. This, in turn, decreases cell–matrix interaction and cell invasiveness, hindering cell migration and metastasis formation. Biological or pharmacological approaches to block TMPRSS4 and ITGα5 may constitute an interesting novel approach to inhibit lung cancer. The authors declare no conflict of interest. We thank the Proteomics and Bioinformatics Unit at CIMA for performing the microarrays and for the analysis of the microarray data. This work has been funded by ‘UTE’ project CIMA, by grants (RD12/0036/0040) from Red Temática de Investigación Cooperativa en Cáncer (RTICC and PI 10/00166), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’, Gobierno de Navarra, and PIUNA (ref 12028402). LL was supported by a Gobierno Vasco fellowship and ALA by Asociación de Amigos (Universidad de Navarra) fellowship. Supplementary Information accompanies this paper on British Journal of Cancer website