Original ArticlesTMPRSS4 induces cancer stem cell-like properties in lung cancer cells and correlates with ALDH expression in NSCLC patients
Introduction
Mortality rates in non-small cell lung cancer (NSCLC) patients remain overly high despite the implementation of novel targeted therapies and chemotherapeutic regimes. The main reason for this problem is that tumors are generally diagnosed in the later stages of the disease and that effectiveness of current treatments is either therapeutically insufficient or only beneficial to a small population of patients (in the case of targeted therapies). Therefore, it is clear that identification of new potential therapeutic targets is a priority for the management of NSCLC.
Metastasis involves a series of phenotypic and behavioral changes in cancer cells that allow them to escape from the primary tumor, migrate through lymphatic and/or blood vessels, survive in circulation and colonize a new organ [1]. In order to be fully metastatic, cancerous cells need to modify their transcriptome to adapt to the new microenvironmental conditions that they will encounter in this multi-step process [1]. Proteases play a major role in tumor progression as they induce the degradation of the connective tissue surrounding the cancerous mass and activate growth factors, cytokines and angiogenic factors that need to be cleaved to be fully active [2]. Type II transmembrane serine proteases (TTSPs) belong to a family of enzymes that includes 18 members, some of which have been demonstrated to promote tumorigenesis, such as hepsin, matriptase, HAT/DESC, TMPRSS2 and TMPRSS4 [3].
TMPRSS4 comprises of a short N-terminal cytoplasmic domain, a transmembrane domain and a large extracellular domain that contains its catalytic activity [4]. This protease is highly upregulated in a variety of solid tumors, including pancreas, colon, lung, ovary, breast, cervical carcinoma and thyroid cancers [4], [5], [6]. We have previously demonstrated that TMPRSS4 is overexpressed in lung tumors compared to normal lung, particularly in squamous cell carcinomas compared to adenocarcinomas [7]. Importantly, high TMPRSS4 levels in patients with squamous cell carcinomas are associated with poor prognosis [7]. We have also shown that the depletion of TMPRSS4 levels using shRNA strategies in NSCLC cells causes significant metastatic impairment in animal models [7].
Mechanistic studies have reported that TMPRSS4 induces epithelial to mesenchymal transition (EMT) and promotes metastasis in colon cancer cells [8], [9]. Through EMT, cells confined within the primary tumor are able to detach from neighboring cells by loss of proteins involved in cell–cell contact, such as E-cadherin, and acquire a migratory behavior [10]. Acquisition of the EMT phenotype by TMPRSS4 is mediated by upregulation of integrin-α5β1 and activation of downstream signaling cascades involving FAK, Src, Rac1, ERK1/2 and AKT phosphorylation [11]. Targeted inhibition of PI3K or Src decreases cell invasiveness and actin rearrangement mediated by TMPRSS4 [9]. Through a microarray analysis, we discovered that miR-205 (MIR205HG) was overexpressed upon TMPRSS4 downregulation [12]. Increased miR-205 levels promote an epithelial phenotype with high E-cadherin and low fibronectin levels, thus inhibiting tumor cell migration and metastasis. Moreover, we identified integrin α5β1 as a new miR-205 direct target in NSCLC [12].
Since EMT favors the dissemination of cancer cells to distant organs, it is thought that this process is necessary for efficient metastasis. In addition, to give rise to new metastatic tumor masses, the property of self-renewal seems to be another required trait. This is because disseminated cells have to act as founders of new cancerous colonies. For these reasons, some authors have hypothesized that migratory cells with an EMT phenotype can share properties with cancer stem cells (CSCs) [13]. Current evidence suggests that CSCs are responsible for tumor initiation, cell survival after therapy, metastatic spread and tumor recurrence [14]. CSCs are resistant to regular chemotherapeutic drugs and to radiotherapy [14], [15]. This suggests that many cancer therapies, while shrinking the tumor bulk, may fail overall because they do not eliminate completely the CSC population.
Based on this information we hypothesized that TMPRSS4, an inducer of EMT, could also confer CSC properties. Therefore, the aim of this study was to assess whether TMPRSS4 induced CSC properties in lung cancer models and whether TMPRSS4+ tumors from NSCLC patients would be associated with a CSC phenotype, supporting its role as a tumor promoting gene. Indeed, we have found that the expression of TMPRSS4 is associated with CSC features including tumorigenicity, formation of primary and secondary tumorspheres (a sign of self-renewal), regulation of the CSC marker ALDH, resistance to regular chemotherapy, and increased sensitivity to the CSC-targeted drug, salinomycin. Moreover, TMPRSS4 expression is very significantly correlated with that of ALDH and OCT4 in patients tumor samples. All these results suggest that TMPRSS4 may induce a cancer stem-like cell phenotype that promotes tumorigenesis.
Section snippets
Cell culture
The human lung cancer cell lines H358, H441, H460 and A549 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A primary cell line obtained from a pleural effusion (designated as Gon8) that we have previously characterized [16] was also used. The Lacun3 cell line, derived from a murine lung adenocarcinoma after injection of DMNA and silica, has been previously described and characterized by our group [17]. All cell lines were grown and cultured in RPMI
Characterization of TMPRSS4-overexpressing A549 cells
Firstly, we wanted to examine the effect of TMPRSS4 overexpression in cells with low endogenous expression of this protein, as is the case for the A549 cell line [7]. Retroviral particles carrying either the empty vector (as controls; designated as pBABE) or the vector carrying TMPRSS4 (referred to as pB-TMP4) were used. TMPRSS4 expression levels in these cells were measured by RT-PCR and Western blot. In addition, we performed immunohistochemistry as a further validation of protein expression.
Discussion
TMPRSS4, a serine protease that is highly up-regulated in several cancer types, has been shown to promote invasion, tumor growth and metastasis [4]. Although the precise mechanism and signaling cascades are not yet well understood, induction of EMT through up-regulation of integrin-α5 seems to be a critical mechanism for its tumorigenic role. Activation of several extracellular factors by TMPRSS4-mediated proteolysis contributes to metastasis. For example the pro-urokinase-type plasminogen
Conflict of interest
We state that no conflict of interests exist in our study.
Acknowledgements
This work has been funded by RTICC (RD12/0036/0040), FIS (PI13/00093) and MINECO (BIO2013-50458-EXP). Arrate L. de Aberasturi was supported by an “Asociacion de Amigos de la Universidad de Navarra” fellowship; Maria Villalba was supported by an FPU fellowship (FPU-14/03247); Stephanie R. Evans was supported by an ERASMUS fellowship.
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IDISNA: Navarra's Health Research Institute.