Epigenetic control of INSL4 promotes cell growth and invasiveness in Non-Small-Cell Lung Cancer


 Background Non-Small Cell Lung Cancer accounts for 80–85% of all forms of Lung Cancer as leading cause of cancer-related death in human. Despite remarkable advances in the diagnosis and therapy of Lung Cancer, no significant improvements have thus far been achieved in terms of patients’ prognosis. Here, we investigated the role of INSL4 – a member of the relaxin family –in NSCLC.Methods We permanently overexpressed INSL4 in NSCLC cells in vitro to analyse the growth rate and the tumourigenic features. We further investigated the signalling pathways engaged in INSL4 overexpressing cells and the tumour growth ability by studying the tumour development in a patient derived tumour xenograft mouse model. Results We found a cell growth promoting effect by INSL4 overexpression in vitro in H1299 cells and in vivo in NOD/SCID mice. Surprisingly, in NSCLC-A549 cells, stable INSL4 overexpression has not showed similar effect, despite has an INSL4-mRNA expressed up to 22.000 fold more respect H1299. The INSL4-mRNA analysis of eight different NSCLC-derived cell lines, has revealed a great discrepancy between the amount of INSL4-mRNA and specific protein. Notably, similar result has been observed in studied NSCLC patients analysing and comparing INSL4 mRNA and protein expression. However, in a cohort of NSCLC patients, we found a significant inverse correlation between INSL4 expression and Overall Survival.Conclusions By combining the results from the in vitro and in vivo models and in silico analysis in patients whose NSCLCs adenocarcinoma spontaneously expressed high levels of INSL4 our results suggest that epigenetic modifications that affect INSL4 does not allow to assess precision therapy in selected patients without consider protein INSL4 amount.

Indeed, at first we compared two different cell lines bearing different mRNA expression of INSL4, who showed a huge discrepancy between INSL4 gene expression and protein amount, later we analysed the divergences in eight different LC cell lines defining that INSL4 mRNA is highly regulated at posttranscriptionally/post-translationally level. Thus, we analysed expressed levels of INSL4 in patients with NSCLCs adenocarcinoma. A significant discrepancy was found to occur between levels of INSL4 mRNA expression and protein content. Moreover, in silico analysis for assessing patients whose NSCLCs adenocarcinoma spontaneously expressed high levels of INSL4, a significant inverse correlation was found to occur between levels of INSL4 expression with poor Overall Survival (OS). By combining the results obtained from the in vitro and in vivo models and patients our results provide evidence for a potentially important role for INSL4 in the biology of adenocarcinoma NSCLC.

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) cleavagesite 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 (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 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 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 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 22x10 3 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  has to be performed to discriminate the mRNA expression and the protein content to carry out specific therapy ( Figure 8 and Figure 9).

Discussion
LC is a heterogeneous and dynamic disease associated with numerous somatic mutations, amplification and deletion in the cell's genome. NSCLC is the most common LC and is responsible for most cancer-related death worldwide (1,5,21,22). Due to the poor clinical outcome, extensive studies have been aiming at clarifying the molecular mechanism involved in NSCLC onset, progression and recurrence, so to identify potentially druggable molecular targets and genes that could be exploited in prediction models for assessing the risk of recurrence. Molecular profile prediction of cancer is, indeed, key to the implementation of personalized therapeutic manoeuvres (7,23,24). Molecular targeted therapies are now being included in treatment regimens for LC patients as they have been shown to extend progression-free survival and improve overall survival (9,25,26).
INSL4 was first discovered in the placenta tissue and belongs in the relaxin/insulin-like family of peptides, which have been credited over the years with a functional role in cancer (12,13,16,(27)(28)(29).
The functional consequences of relaxin receptor activation in cancer cells include increased cell motility (16) and tumour growth and angiogenesis (27,30), all of which contribute to tumour expansion, tissue invasion and metastasis formation.
Here we demonstrate that INSL4 is an active tumour-promoting gene in NSCLC, in that it favours proliferation, invasion and migration of LC cells. INSL4 overexpression increased mitosis in NSCLC H1299 cells and promoted cell-cycle progression. For the first time the biological relevance of INSL4 was substantiated in assays of colony-formation ability and migration. In particular, we observed that overexpression of INSL4 provides cells with an ability to generate colonies with higher cellular density, suggestive of loss of cell-contact inhibition and increased proliferation. On assaying INSL4overexpressing cells in soft agar, we found that INSL4 affects the ability of transformed cells to acquire anchorage-independent growth (31,32). Of note, INSL4 overexpression will also affect the original morphology and cytoskeleton arrangements in NSCLC H1299 cells, resulting in the appearance of large and numerous protrusions.
We also demonstrated that INSL4 autocrine and/or paracrine effects involve the activation and/or enhancement of the MAPK and AKT signalling pathways. Genetic mutations or Gain-Of-Function events can deregulate or hyperactivate the MAPK and/or AKT pathways during induction and progression of tumorigenesis. In particular, their activation is known to be key to LC development (17,18,33,34). INSL4 could thus promote cell proliferation and invasiveness, by upregulating the MAPK and AKT signalling pathways, respectively. AKT activation is recognized as an important factor contributing to invasiveness of cancer cells (34,35). Several studies have reported a crosstalk between the MAPK and AKT signalling pathways in order to sustain tumour cell proliferation (17,18,35). All the experiments were performed in triplicate at least three times.

RNA extraction and Quantitative Real-Time PCR (qPCR)
The study protocol was approved by the local Ethics Committee and was conducted in accordance with the ethical principles of the latest version of the Declaration of Helsinki. Written informed consent was obtained from all patients before enrolment. Tumour tissue specimens were collected from patients diagnosed with AC-NSCLC. RNA extraction and qPCR were performed as previously described (38). Total RNA was extracted from tumour tissues using TRIzol® (Invitrogen), according to the manufacturer's instructions. cDNA was retrotranscribed from 1 µg of RNA using the iScript kit  Experiments were performed at least three times and performed each time in triplicate.

Colony formation assay
For Colony formation assay cells were seeded at 5 × 10 2 in 6-well dishes and the following day, complete medium was replaced with RPMI 1640 + 5% FBS and cells were grown for 2 weeks. At the endpoint, plates were fixed with 4% formaldehyde (Sigma-Aldrich) and stained with 2% crystal violet (Sigma-Aldrich). Finally, the plates were used for image acquisition with a digital camera. Experiments were performed at least three times and performed each time in triplicate.

Soft agar colony formation assay
Anchorage-independent growth of tumour cells was estimated by a soft agar colony formation assay.
In brief, 1,2% base agar in RPMI-1640 complete medium was polymerized in 6 wells Petri dishes to make a growth substrate. Cells (5 × 10 3 /well) were quickly resuspended in agarose (1%) in complete medium and the mix was carefully stratified onto the base agar. Foci were allowed to develop for a 10-14-day incubation. Twenty fields were selected for each cell condition to count Colony Forming Tumour size was measured with a caliper each 5 days and volumes were estimated using the formula: length (mm) x width (mm) x height (mm)/2. Animals were sacrificed 4 weeks after tumour cell implantation and tumour was removed, weighed, and split into 2 parts, one to be fixed in 10% buffered formalin for histological analysis, and the other to be snap-frozen in liquid nitrogen and stored at -80 °C for molecular analysis.

Immunohistochemistry
Histological analysis was performed as previously described (39). Briefly, paraffin-embedded tumour tissues were sectioned (4µ m) and stained by haematoxylin and eosin (H&E), to observe tissue structure and calculate mitotic index values by enumerating proliferating cells. For the Ki67 index, the tissue section slides were blocked and incubated with specific anti-Ki67 antibody (Sigma-Aldrich).

Tissue Microarray
Tissue microarrays were prepared as previously described (40). Overall, 2-mm cores were obtained from the most representative areas of the tumours and then re-embedded in microarray blocks. Each case was sampled 3 times. Prior to tissue microarray construction, a H&E slide of each block was analysed to exclude non-representative inappropriate regions (e.g. necrosis and haemorrhage).

Consent for publication
Not applicable

Availability of data and material
The dataset(s) supporting the findings of this study are included within the article and its supplementary information. Further details are available from the corresponding author on request.

Competing Interests
The authors declare no competing financial interests            Stored mRNA builds a cellular reservoir ready to be rapidly recruited to protein translation.

Figure 9
Mechanism of translational control of INSL4. INSL4 mRNA is transcribed either to be (immediately) transduced in INSL4 protein or bound to miRNA. Once engaged in miRNA binding mRNA is driven to degradation or reversible storage into cytoplasmic particles.
Stored mRNA builds a cellular reservoir ready to be rapidly recruited to protein translation.

Supplementary Files
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