INSL4 structure analysis and localization in NSCLC
INSL4 gene was located on human chromosome 9p24, with an open reading frame (ORF) of 417 bp coding for a 139 amino-acid protein (13, 14). Using UniPRO and ExPASy-PROSITE protein databases, we attempted to define INSL4 protein domains. We found a Signal-Peptide Peptidase (SPP) cleavage-site located at the N-terminus and an Insulin Family Signature (IFS) at the C-terminus (Figure 1a, left). Cleavage by SPP was found to occur between amino acids at positions 25 and 26 (Figure 1a, right). The in silico study to determine INSL4 putative localization revealed that INSL4 is a secretory protein mostly restricted to the extracellular compartment. This suggested that INSL4 is cleaved and modified in the Golgi apparatus, where it acquires secretory protein features. Indeed, a short putative Nuclear Localization Site (NLS) has been described between Aa 94-116.
We studied INSL4 localization in H1299 cells by cloning INSL4 in an expression vector, so to generate a fusion protein with the Myc epitope sited in the protein C-terminus. On immunofluorescence analysis, INSL4 was not detectable across the whole cell body, but rather restricted to the Golgi and Endoplasmic Reticulum (ER) compartments, as revealed by immunolocalization with specific antibody and calreticulin respectively, the latter being used as a marker of subcellular district (Figure 1b). To check for any possible changes in intra and extracellular INSL4 localization, as suggested by the in silico analysis of INSL4, we examined H1299 cells for INSL4 expression at 48 and 72 hours of transfection. The longer term at 72 hours provided evidence by immunofluorescence images for the presence of INSL4 in vesicles and granules of secretion (Figure 1b). We thus analysed INSL4 localization in not permeabilized cells at 72 hours of transfection. INSL4-transfected cells were characterized by the obvious presence of INSL4 in the peri- and extra-cellular regions (Figure 1c). Moreover, staining the actin cytoskeleton with phalloidin-FITC conjugate at 72 hours of transfection revealed INSL4 as being localized in actin-rich membrane protrusions (Figure 1d). These results substantiated the data, predicted by the in silico observations, being likewise in accordance with previous data on INSL4 secretion and its distribution in autocrine compartments (12, 15, 16).
INSL4 promotes proliferation and invasiveness by NSCLC
To study the functional significance of INSL4 in NSCLC, we generated INSL4 stably overexpressing H1299 cells (hereafter referred to as H1299-INSL4). As a control, H1299 cells were transfected with the empty vector (Control) (Supplementary Figure S1a). Growth rates were simultaneously observed. At 96 hours of culturing, an increase approximately 27% was found in the proliferative index of H1299-INSL4 cells relative to controls (P < 0.001) (Figure 2a). Next, we analysed mitotic indexes by enumerating mitoses in H1299-INSL4 and H1299-control cells. In INSL4-overexpressing cells, the mitotic index increased by 2.5-fold value relative to controls (Figure 2b). Consistently, cell cycle progression observed by FACS analysis showed that INSL4 promotes a reduction of cells in G0/G1 phase and an increase of those in S phase (Figure 2c).
To further substantiate the effect of INSL4 on cell proliferation, we investigated the behaviour of INSL4 overexpressing cells, in a colony-forming assay. Clonogenic assays were performed using H1299 cells as control and H1299-INSL4. Cells were cultured for 2 weeks under low-serum conditions before assessing their ability to generate colonies. Significant differences in growth rates of H1299-INSL4 compared to H1299 cells were observed. INSL4 overexpression conferred an increased ability on transfected cells to generate colonies with enhanced cellular density relative to controls (Figure 2d). To better document the impact of INSL4 on cell proliferation, we performed a soft agar assay using H1299-INSL4 and H1299-control cells. INSL4 overexpression significantly increased the ability of H1299 cells to form colonies in soft agar (Fig. 2e). Indeed, the analysis of the soft-agar plates showed not only a marked increase in the total number of colonies generated by INSL4, but those overexpressing INSL4 were characterized by a more consistent and fringed pattern relative to controls (Figure 2e).
Another important finding from fluorescent-microscope analysis of H1299-INSL4 cells stained with phalloidin was the observation that INSL4 overexpression modifies cell morphology. H1299-INSL4 cells displayed large and numerous protrusions not found in control cells. Moreover, the protrusions of cellular edges appeared thin and long (Figure 2f). This data strongly suggested that INSL4 increases invasiveness in NSCLC. We asked whether those changes were accompanied by changes in cell motility. Scratch assays were performed in vitro to examine the effects of cell-matrix and cell-cell interactions on cell migration. Comparative analysis of H1299-INSL4 and control cells showed a clear difference in motility patterns between the two populations, indicating INSL4 may indeed increase NSCLC motility (Figure 2g).
Because the release of INSL4 from cells could have autocrine and paracrine effects, we studied the pathways exploited by the insulin-like factor to activate cell growth. Although a class of receptors have been identified for INSL peptides, namely, the RXFP receptor family, the specific nature of the receptor for INSL4 has yet to be defined. We investigated the ability of INSL4 to trigger the MAPK pathway, one of the most important signalling pathway involved in cell proliferation (17, 18). Western blotting analysis of H1299 as control and H1299-INSL4 cells revealed an increased MAPK phosphorylation in the INSL4 overexpressing population. Similar phenotype was observed analysing AKT phosphorylation (Figure 2h). The AKT signalling pathway plays a central role in many cellular processes, and it contributes to cancer progression (19).
INSL4 overexpression leads to increased tumour growth in vivo
Because INSL4 has a functional in vitro effect in NSCLC proliferation and invasion, we investigated any contributions of INSL4 to tumour growth in vivo. H1299 as control and H1299-INSL4 were injected into NOD/SCID mice to study the proliferative potential of the cell lines. Tumours size was measured at 5 day intervals after injection and all mice were sacrificed on day 25 after engraftment. Mouse grafting with H1299-INSL4 cells resulted in a 2-fold increase in growth rates relative to control xenografts, as demonstrated by tumour growth kinetics. Tumour xenografts of both cell types showed similar growth patterns until day 10, when a difference became instead appreciable. Endpoint analyses of tumour weights showed a 50% increase in tumour masses of H1299-INSL4 recipient, relative to control mice (Figure 3a). Tumour growth was indeed obvious on gross inspection of H1299-INSL4 tumour-bearing hosts, with histopathology showing extensive vascularization as well as infiltration of muscular tissues and bones in rib cages. Histopathology further revealed poorly differentiated H1299-INSL4 cells, whose morphology was marked by the widespread presence of mitoses. On enumerating mitotic cells in both types of tumour, a 2.7-fold increase was found in INSL4-overexpressing masses (Figure 3b). Moreover, the H1299-INSL4 tumour masses present necrotic areas, which were instead absent in control specimens (Figure 3c). A 2-fold higher labelling index of Ki67 – a hallmark of cell proliferation – was found in the latter cells (Figure 3d). Likewise, in the in vitro setting, we detected MAPK and AKT activation in extracts from INSL4-overexpressing tumour masses (Figure 3e).
To verify the important role of INSL4 in LC, we further analysed, the A549 cell line. We stably transfected A549 cells with the fusion protein INSL4-myc-Tag, (hereafter referred to as A549-INSL4) and the empty vector as control (Control). The INSL4 expression was controlled by Western Blotting analysis (Supplementary Figure S1b) and growth rate was assessed. Unexpectedly, at 72 hour of culturing, a decrease in the proliferative index around of 50% was found in A549-INSL4 cells relative to controls (Figure 4a).
After this unpredicted result, we investigated other proliferative indicators in A549-INSL4 cells such as colony-forming assay and soft agar. Clonogenic assay was performed in A549-INSL4 and A549 used as control cells. As expected, no significant differences in growth rates of A549-INSL4 compared to A549 cells were observed (Figure 4b). Then, we performed soft agar assay in A549-INSL4 and A549 cells. Also in this case, INSL4 overexpression did not increase the number and the size of colonies in soft agar (Figure 4c).
After these results, we asked why this discrepancy in growth rate between H1299 and A549 was present. Both cell lines selected showed an increased in INSL4 protein after transfection (Supplementary Figure S1b). Indeed, comparing the basal mRNA expressed in the H1299 and A549, we found that INSL4 mRNA is highly expressed in A549, almost 22x103 times respect to H1299 (Figure 5a left). At this point, we analysed INSL4 protein levels in untreated H1299 and A549 and remarkably we found that a huge discrepancy is present between the amount of mRNA and protein in the two cell lines. In particular, in H1299 the amount in protein is little less respect to A549, despite in this last, the mRNA is much more expressed (Figure 5a right). To determine whether the control in translation of INSL4 mRNA is distinctive of A549 cell lines or it is present in other tumour cell lines, we analysed eight different tumour cell lines of NSCLC. We explored the mRNA content of INSL4 in: H1299, A549, H1650, H1975, H460, HCC827, CALU-3 and CALU-1 assuming the amount of INSL4 mRNA of H1299 as Arbitrary Unit 1. The data obtained show that in three cell lines on eight, in particular A549, H460 and CALU-3 we found a huge increase in INSL4 mRNA with values of 22262, 7882 and 23198 respectively compared to H1299 (Figure 5b left). Then we evaluated INSL4 protein levels of all the cell lines examined. Notably, although the high differences in mRNA amount, the protein content in all the LC cell lines showed little differences among them (Figure 5b right). This data highlighted a great discrepancy between INSL4 mRNA and relative protein expression.
Because the divergence between transcription and translation of INSL4, we analysed the stability of INSL4 protein in H1299 and A549 cell lines. We exposed the two cell lines to cycloheximide and analysed the cells at different times after treatment. No difference in INSL4 protein stability has been detected in H1299 and A549 (Figure 5c and 5d).
INSL4 analysis in NSCLC patients
Because the great difference detected in LC cell lines examined, we compared mRNA expression and protein content in a cohort of patients with NSCLC. In accordance with previously reported gene expression profiles, we examined a cohort of patients with AC-NSCLC (10). INSL4 mRNA expression of eight individual patients was determined by qPCR analysis. INSL4 mRNA levels were significantly up-regulated in four NSCLC patients out of the eight being examined, relative to normal lung tissues from healthy subjects (Figure 6a).
Importantly, the histological analysis of the specimens shows similar results to those observed in LC cell lines. In patients with NCLSC we observed that a high expression of INSL4 mRNA does not always correlate with a high amount of protein, while in some patients with low INSL4 mRNA a great INSL4 protein content is present (Figure 6b). These data indicate that there is no linear correlation between expression of the INSL4 gene and protein, but an individual epigenetic control in each single patient should be strongly considered.
INSL4 overexpression predicts poor survival in NSCLC patients
As previously described INSL4 is expressed in adult life in placenta and is expressed again in some tumours. To substantiate a clinically relevant tumourigenic role for INSL4, we used cancer outlier profile analysis as applied to the web-accessible GENT2 microarray database, containing samples from the Affymetrix U133Plus 2.0 platform. INSL4 expression assessed in a set of different cancer histotypes revealed remarkable INSL4 expression mostly occurring in LC tissues relative to control counterparts (Figure 7a and 7b). By extrapolating the analysis of a cohort of 2362 NSCLC patients and 508 normal lung samples, we observed a large prevalence of patients with INSL4 overexpression (Figure 7b). Screening of the COSMIC data bank to evaluate INSL4 overexpression in LC patients indicates that INSL4 is overexpressed in almost 4% of all screened NSCLC patients. These results were in agreement with the findings above and reinforced the notion that INSL4 is a potential oncogene (20).
To elucidate the association of INSL4 expression with clinical endpoints in NSCLC patients, we used the Kaplan Meier Plotter. In all NSCLC patients, high INSL4 expression was significantly associated with shortened Overall Survival (OS, P=0.00094, HR=1.24) as well as with reduced First Progression (FP, P=6.8e-08, HR=1.7) and Post Progression Survival (PPS, P=0.017, HR=1.36) (Figure 7c). Of note, in patients with AC-NSCLC, increased INSL4 expression was significantly associated with poorer OS (P=0.0019, HR=1.44). A similar correlation was observed between INSL4 status and PPS in AC-NSCLC patients (P=0.0018, HR=1.75) (Figure 7d). In contrast, there occurred no significant association between INSL4 expression and clinical outcomes in patients with the squamous cell carcinoma-NSCLC (Figure 7e). Therefore, INSL4 appears to be a specific marker of poor prognosis in patients with AC-NSCLC. These data confirm an important role played by INSL4 in LC, but a selection in each patient has to be performed to discriminate the mRNA expression and the protein content to carry out specific therapy (Figure 8 and Figure 9).