Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.


Introduction
Lung cancer is the first cause of neoplastic death worldwide in both men and women population [1]. Non-small cell lung carcinoma (NSCLC) accounts for 80% of all cases. Nevertheless the recent progress in NSCLC therapy with the advent of personalized medicine, only 18% all patients are still alive after 5 years from diagnosis [2]. In lung cancer, Vitamin A (retinol) deficiency has been associated with bronchial metaplasia and increased cancer development [3][4][5]. Retinoids (retinol derivatives) showed to promote differentiation and cell death of cancer cells in a number of experimental systems, including lung [5][6][7]. In recent years, new treatments with novel action mechanisms have been explored for advanced lung cancer, including retinoids administration [3,7,8]. Biological activity of retinoids, in particular all-trans retinoic acid (atRA) is normally mediated by specific cytoplasmic and nuclear receptors [8][9][10]. Cellular retinol binding protein-1 (CRBP-1) is a 15 kDa cytosolic binding protein crucial for uptake and subsequent esterification of retinol, by regulating its bioavailability [8,11]. CRBP-1 is indispensable for embryonic development and growth of vertebrates. In the lung parenchyma, CRBP-1 expressing cells are observed during development and prenatal alveolus formation [12]. Defects in CRBP-1 gene expression linked to oncogenic process in breast, prostatic, renal, lung and endometrial cancer [13][14][15]. Retinoids exert their pleiotropic and transcriptional effects binding nuclear receptors, namely the retinoic acid receptors RARα, β, and γ, and retinoid X receptors RXRα, β, and γ [5,8,10,16]. RARs may form homodimers [17,18] or heterodimers with other receptors as thyroid hormone receptors, Vitamin D3 receptors, peroxisome proliferator active receptors (PPARs) and several orphan receptors, contributing to starting alternative signalling pathways [19]. For example, RARβ and RXRα, in normal respiratory epithelium, binding PPARγ with other cofactors, ensures cyclin D1 mediated cell cycle inhibition hence favouring apoptosis or differentiation [5,18]. Down regulation of RARβ combined with AP-1 upregulation triggers tumour progression and proliferation of NSCLC cells [20]. Concurrently, the inability of RXRα to form heterodimers with PPARγ enables an AP-1/CRBdependent up regulation of Cox2, resulting in inhibition of apoptosis [16,21]. The loss of RARβ mRNA expression has been observed in many lung cancer cells line and its expression is contingent on intracellular concentration of retinoids, mediated by CRBP-1 and 2 [20]. Moreover, retinoids can activate several pathway, including AKT/ERK signalling in lung cancer cells through a transcriptional independentmechanism [3,22]. Retinol can induce cytokine-dependent activation of Jak2 and subsequently STATs transcription factor, while the exchange from RBP to intracellular CRBP-1 is mediated by Stra6 receptor [23].
We recently documented that high expression of CRBP-1 in lung adenocarcinoma in vivo was associated to a lower overall survival [24]. Napoli et al. proposed that CRBP-1 expression in NSCLC should be considered as marker of RARβ down regulation [8]. In fact, authors suggested that CRBP-1 cytoplasmic accumulation represent a block of nuclear availability of retinoids.
In this preliminary study, we investigated the effect of atRA in native and CRBP-1-transfected H460 lung cancer cells with particular reference to the modulation of RAR/ RXRs and pAKT/pERK/pEGFR gene signaling. We highlighted that the restoration of CRBP-1 influenced the proliferation and AKT signalling pathway in H460 cell line also in the presence of atRA treatment.

Cell transfection
Human non-small cell lung cancer H460 cell line (kindly provided by Dr. Carlo Leonetti, Regina Elena National Cancer Institute, Rome, Italy) maintained in RPMI 1640 (Lonza Bio Pharma AG, Switzerland) and transfected by using a vector pTargeT. Mammalian expression system carrying the whole sequence of CRBP-1 gene (NM_002899) and the gene for the resistance to G418 (Promega, Italy), or the G418-resistance gene alone, as reported [25]. After 20 days, stable transfected clones were collected in G418-containing medium and tested by PCR and western blot. The correct plasmid sequence confirmed by Sanger sequencing. Experimental procedures were repeated by using different transfected clones, which gave similar results (data not shown).

Gene expression analysis
Gene expression analysis was performed using Signosis array (Signosis, Inc., Santa Clara, CA, USA). Briefly, total RNA was extracted [27,28], reverse-transcribed into cDNA in the presence of biotin-dUTP and a profile of 24 genes for human AKT and Stat-3 pathway cDNA plate array (Catalog number AP-0161 and AP-0151 respectively; Signosis). Luminescence relative light units (RLUs) was evaluated on a microplate luminometer, according to the manufacturer' instruction. Real-time PCR for RXR, RARs and cytokeratins was also performed in triplicate using β2-microglobulin, β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping genes, as previously reported [24].

Patients and methods
Paraffin blocks of tumors of a small cohort of patients with histologic diagnosis of NSCLC (Santa Maria della Misericordia Hospital of Perugia, Italy) were included. Tumor classification was in accordance with WHO criteria and the most diffuse immunohistochemical panel. For CRBP-1 positive and negative expression, tissue sections were incubated for 1 h with rabbit polyclonal anti-CRBP-1 (1:200; clone FL-135, Santa Cruz Biotechnology, Heidelberg, Germany). Diaminobenzidine was used as final chromogen. CRBP-1 expression was estimated at × 400 magnification by two of the Authors by using the following semi-quantitative grading system: absent and strongly positive expression. This retrospective study was approved by Ethics Committee, waiving patient consent.

Statistical analysis
Results were analysed as the arithmetical mean ± SD Data were analyzed by one-way analysis of variance (ANOVA) followed from a Bonferroni post hoc test or using the Student t-test. The differences were considered statistically significant for p values < 0.05. All the statistical analyses performed with SSPS V.20 (Stat Corp, College Station, TX, USA).

CRBP-1 expression influences AKT signaling pathway in vivo
To further investigate if CRBP-1 positive expression could modulate AKT pathway in vivo, a preliminary gene array was performed. One patient with CRBP-1 + tumor and one patient with CRBP-1 − tumor expression were analysed. As reported in Fig. 4, genes implicated in AKT pathway resulted down-regulated, whereas only PTEN was up-regulated.

Discussion
The H460 cell line represents a suitable experimental model of highly proliferative, drug-resistant, metastatic NSCLC cell line [7]. Recently, it has been demonstrated the presence of cancer stem cells in H460 cell line [31] thus explaining at least in part its aggressiveness. It is well documented that chemio and radiotherapy resistance is mainly due to so-called cancer stem cells selection, epigenetic mechanisms silencing physiological pathway (for example hypo or hypermethilation of genes) and finally the acquisition of mutations [32]. In fact, also with the promising tyrosine kinase inhibitors or the immunotherapy patients develop inevitably progression [28,33,34]. AtRA is well known to have a dramatic effect on M3 subtype of acute myeloid leukemia [34]. However, in most other cancers this effect was not observed because of epigenetic silencing of retinoid pathway [35]. Low doses of atRA (20 mg/m 2 /day) in combination with chemotherapy showed remarkable activity as demonstrated in a randomized phase II trial of patients  , RARs/RXR (c), pAKT/AKT, pERK1/2 and pEGFR/EGFR (d) protein expression in CRBP-1 + compared to empty-tranfected H460 cells maintained with 10% FBS; e-g Bar graphs of pAKT/AKT, pEGFR/EGFR and pERK1/2 protein expression in CRBP-1 + compared to empty-tranfected H460 cells after 48 h of treatment with 5 µM atRA and ROL. *p < 0.05, **p < 0.005 and ***p < 0.001. RLU relative light unit, ADU arbitrary densitometric units with advanced NSCLC [6]. Our results showed a reduced viability and the down-regulation of several genes and proteins involved in proliferative and transcriptional pathways in CRBP-1 + compared to empty-transfected H460 cells in both basal condition and after atRA treatment. AtRA is often used in clinical trials to suppress the growth and progression of different cancer types [18,36]. However, its effectiveness is limited in some cancer, including lung cancer [6,37,38]. AtRA is an active metabolite of Vitamin A that regulates diverse cellular functions such differentiation, proliferation and apoptosis by binding with RAR/RXR receptors [8]. RARβ is a tumor suppressor gene whose expression is significantly decreased in human cancers and increased with atRA treatment [39]. The loss of RARβ mRNA expression has been observed in many lung cancer cells lines and its expression is influenced from intracellular concentration of retinoids, mediated by CRBP-1 [20]. Besides trafficking, CRBP-1 loss is responsible for intracellular retinoid deficiency, since CRBP-1 is required for retinol bioconversion [40][41][42]. Epidemiological studies suggested that the addition of atRA or synthetic retinoids to human cancer cell lines or human tumor xenografts in nude mice results in a cell growth arrest, apoptosis or differentiation [43]. Expression of CRBP-1 may help to switch between a proliferative and differentiative phenotype in response to oncogenetic stimuli [44]. It has been reported that in human mammary tumor cells the reintroduction of CRBP-1 reduces tumorigenicity in athymic mice [45]. Moreover, the inhibition of PI3K/Akt pathway was involved in the antitumor effect of CRBP-1 mediated from p85 regulatory and p110 catalytic subunit heterodimerization [46]. It is possible to hypothesize that re-expression of PI3K/Akt signaling mediates CRBP-1 loss-induced cancer progression and RARβ down-regulation in a transcription-independent mechanism of action of atRA.
Several in vitro studies showed that atRA induces a transcription-independent activation of the PI3K/Akt pathway [18,24]. For that reason, we transfected H460 lung cancer cell line with CRBP-1 and compared the effect of atRA in Akt signalling and RAR expression. We observed that restoration of CRBP-1 expression in H460 cells reduces survival and clonogenicity compared to emptytransfected cells. Moreover, CRBP-1 + H460 cells showed a poor ability to form colonies compared to CRBP-1 − cells, both in basal condition and after 48 h of atRA treatment.
A series of genes involved in Akt and Stat-3 related proliferation pathways were down-regulated in CRBP-1 + H460 cells as Akt1, Akt2, Akt3, Foxo3, p21, p27, p70, Casp9, Gsk-3a, IGF-1R, PI3K, C-myc, Cycline-E, whereas, Bcl-2, GP130, Bcl-xl, GSK-3B, C-fos genes were up-regulated. Those findings confirmed the hypothesis that CRBP-1 expression in H460 cells reduces the expression of proliferative and increases that of differentiation markers. As concerning cytokeratin expression, we observed the upregulation of cytokeratin-1 and 10 and the downregulation in cytokeratin-5 expression in CRBP-1 + compared to empty-transfected H460 cells. In basal condition, CRBP-1 + H460 cells showed a significant reduction of pAKT protein expression, but no significant difference in pERK and pEGFR expression. A greater and significant downregulation of pAKT, pERK and pEGFR in CRBP-1 + compared to empty-transfected H460 cells was documented after 48 h of 5 µM atRA treatment, but not after retinol treatment. As possible explanation, it has been reported that genes related to retinoid biosynthesis, transport, degradation and signaling are deregulated in most NSCLC [47], possibly explaining at least in part those contradictory results. In particular, retinol is transformed in retinal through many enzymes, all of which may be inactivated or non-functioning [47]. Finally, we showed that RARα expression was decreased and RARβ increased in CRBP-1 + where compared to empty-transfected H460 cells. Therefore, CRBP-1 restoration influenced proliferation and AKT signaling pathway in H460 cell line also in the presence of atRA treatment. Both atRA and retinol were dissolved in DMSO, which acts as a histone deacetylase inhibitor. The DMSO action could be a limitation in our study. Nevertheless, being used for the dissolution of both drugs, the differences reported cannot be due to the presence of DMSO. Finally, further studies are needed to define the atRA role in CRBP-1 + H460 cell line and to clarify if those CRBP-1-related pathways involved in NSCLC carcinogenesis may be modulated, with a potential beneficial opportunity for a more personalized chemotherapeutic regimens employing adjuvant retinoid therapy.