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Tumour necrosis factor-α (TNF-α) is a double-edged cytokine associated with pathogenesis of inflammatory-related cancers being also able to induce cancer cell death. In the process of tumour development or metastasis, cancer cells can become resistant to TNF-α. In trefoil factor 3 (TFF3) overexpressing colorectal adenocarcinoma cells (HT-29/B6), we observed enhanced resistance against TNF-α/interferon gamma-induced apoptosis. TFF3 is a secreted small peptide that supports intestinal tissue repair but is also involved in intestinal tumour progression and scattering. We hypothesised that TFF3 rescues intestinal epithelial cancer cells from TNF-α-induced apoptosis by involving regulatory RNA networks. In silico-based expression analysis revealed TFF3-mediated regulation of selected microRNAs as well as long non-coding RNAs (lncRNAs), whereas miR-491-5p was identified to target the lncRNA ‘psoriasis susceptibility-related RNA gene induced by stress’ (PRINS). RNA interference-based gain- and loss-of-function experiments examined miR-491-PRINS axis to exert the TFF3-mediated phenotype. Chemical inhibition of selected pathways showed that phosphatidylinositol 3-kinase/AKT accounts for TFF3-mediated downregulation of miR-491-5p and accumulation of PRINS. Moreover, we showed that PRINS colocalises with PMAIP1 (NOXA) in nuclei of HT-29/B6 possessing inhibitory effects. Immunoprecipitation experiments proved molecular interaction of PMAIP1 with PRINS. Our study provides an insight into RNA regulatory networks that determine resistance of colorectal cancer cells to apoptosis.
Impaired mucosal repair and consequently affected intestinal homeostasis seem to be the cause for a variety of intestinal diseases. Modulating effects on intestinal homeostasis and disease have been reported for human trefoil factors (TFFs). So far, three TFFs have been identified by the presence of so-called trefoil factor domains. The intestinal trefoil factor (ITF or TFF3) is known to act as a driving factor of intestinal epithelial repair.1 In a TNBS-induced murine colitis model, TFF3 has been shown to decrease tissue levels of tumour necrosis factor-α (TNF-α) suggesting a therapeutic potential of TFF3 in inflammatory bowel disease.2 Accordingly, TFF3−/− mice have been shown to develop increased intestinal apoptosis. By contrast, increased expression of TFF3 has been also detected in tumours, where it appears to act as a potent mitogen and inducer of epithelial migration associated with tumour invasion, resistance to apoptosis and metastasis.3 Furthermore, it has been shown that TFF3 prevents TP53-dependent apoptosis of gastrointestinal cancer cell lines in a phosphatidylinositol 3-kinase (PI3K) dependent manner.4 Overexpression of TFF3 significantly inhibits IL-1β induction of TNF-α, this effect was reversed after pre-treatment with the PI3K/AKT inhibitor LY294002.5
The superfamily of TNF-proteins has pleiotropic effects.6 TNF-α-related apoptosis-inducing ligand (TRAIL) activates the extrinsic apoptosis pathway in a variety of tumour cells but not in normal cells.7 Interferon gamma (IFN-γ) has a promising role in tumour suppression by enhancing TRAIL-induced apoptosis.8 Moreover, a combination of IFN-γ and TNF-α has been reported to markedly sensitise metastatic colon carcinoma cells to TRAIL-induced apoptosis.9 Increased TRAIL receptor expression along with activation of the Wnt/β-catenin pathway has been associated with aggravated survival of patients with colorectal carcinoma. At the same time, increased β-catenin has been shown to gradually increase TRAIL receptors.10 Interestingly, TFF3 activates AKT by EGFR phosphorylation, which inhibits ubiquitination of β-catenin and consequently causes its nuclear translocation to promote proliferation of colorectal carcinoma cells.11 On a different note, Shiah et al.12 have shown that non-coding RNAs (ncRNAs) regulate the activator of Wnt/β-catenin signalling Wnt-7b. The tumour-suppressing long non-coding RNA (lncRNA) MEG3 and microRNAs (miRNAs) miR-329 and miR-410 have been reported to be downregulated in oral squamous cell carcinoma resulting in upregulation of Wnt-7b and β-catenin signalling.
Recent efforts in RNA biology have revealed that ncRNAs control central cellular processes such as apoptosis in gastrointestinal malignancies.13 Only about 10% of human RNAs are translated into functional proteins. The rest is classified into the category of ncRNAs such as the well-characterised class of miRNA or the rather heterogeneous class of lncRNAs. MiRNAs regulate gene expression via degradation of mRNAs or inhibition of translation taking part in central cellular processes.14 For example, direct interaction of miR-491-5p with BCL2L1 has been reported to induce apoptosis in colorectal cancer cells.15 Interestingly, miR-491-5p transfected hepatoma-derived Huh7 cells have been shown to suppress phosphorylation of AKT pointing to inhibition of PI3K-pathway.16 But also interactions between miRNAs and lncRNAs have been described, prompting the idea of regulatory networks of RNAs.17 Interactions between ncRNAs are suggested to provide a plethora of unprecedented regulatory mechanisms controlling development and disease. LncRNAs have been reported to be involved in several steps of cancer development as essential regulators, oncogenes or tumour suppressors.18,19 Along these lines, we have recently shown that the lncRNA MEG3 is a regulator of autophagy in macrophages.20 Another lncRNA, psoriasis susceptibility-related RNA gene induced by stress (PRINS) has been related to proliferation and differentiation states of keratinocytes. Increased expression has been described in healthy epidermis compared with psoriatic lesions.21 Silencing of PRINS by RNA interference (RNAi) has revealed its protective role in keratinocytes exposed to stress.22
TFFs are pro-invasive scatter factors interfering with PI3K/AKT, EGFR and Wnt/β-catenin signalling.23 There is increasing evidence that these pathways crosstalk in progression of cancer.24 In addition, several studies point out the involvement of ncRNAs in regulation of mentioned TFF-related pathways. Therefore, we speculated that TFF3 exerts its protective effects on scattering cancer cells via regulatory ncRNAs. To address this, we used TFF3-overexpressing colorectal adenocarcinoma cells (HT-29/B6/TFF3) as a model for scattering cancerous cells that are protected from IFN-γ/TNF-α-induced apoptosis. We here show that the protective phenotype is based on the interaction of miR-491-5p with the lncRNA PRINS thereby regulating the availability of the pro-apoptotic factor PMAIP1 (NOXA).