Colorectal Cancer Aggressiveness is Related to Fibronectin Over Expression , Driving the Activation of SDF-1 : CXCR 4 Axis

C l i n M e d International Library Citation: Gouveia-Fernandes S, Carvalho T, Domingues G, Bordeira-Carriço R, Sérgio Dias, et al. (2016) Colorectal Cancer Aggressiveness is Related to Fibronectin Over Expression, Driving the Activation of SDF-1:CXCR4 Axis. Int J Cancer Clin Res 3:072 Received: August 12, 2016: Accepted: November 16, 2016: Published: November 19, 2016 Copyright: © 2016 Gouveia-Fernandes S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Gouveia-Fernandes et al. Int J Cancer Clin Res 2016, 3:072


Introduction
Cancer hallmarks result from dynamical interactions between tumor cells and tumor microenvironment, including normal cells, growth factors and extracellular matrix (ECM) components [1].Fibronectin (FN) is a high molecular weight, multidomain glycoprotein [2,3], present as a soluble form in body fluids, such as plasma (plasma FN), and as an insoluble form in ECM (cellular FN) [2,4,5].FN exists, generally, as a dimer, composed of two nearly identical polypeptide chains covalently linked by a pair of C-terminal disulfide bonds.This glycoprotein has been implicated in a wide variety of cell functions, particularly those involving 19503-Qiagen) and cDNA was synthesized from 1 µg of RNA, using Superscript II ® (CatNo-18064-014Invitrogen), according to manufacturer's protocols.

Construction of pcdna3.1 -full-length fibronectin (pcdna3flfn)
Full-length FN (embryonic) was amplified from cDNA synthesized from SW480 cells (ATCC, CCL-228™) RNA.cDNA synthesis was performed as described above, but a panel of 23 reverse primers of 15 mer (Supplementary Table 1) was used instead of random primers, to ensure the specificity and enrichment of cDNA from FN; cDNA synthesis occurred for 4 hours.PCR was performed by using an Expand Long Template PCR System (CatNo-11681834001-Roche), according to manufacturer's instructions.Primers were designed as instructed by pcDNA3.1 Directional TOPO Expression kit protocol (CatNo-K4900-40-Invitrogen) (Supplementary Table 1).
The amplified product was visualized in a 1.2% (w/v) agarose gel electrophoresis with 0.005% (v/v) ethidium bromide.DNA fragment with the expected size was extracted using a QIAquick Gel Extraction kit (CatNo-2870-Qiagen), according to manufacturer's protocol.pcDNA3-flFN expression vector was constructed by using the pcDNA™3.1 Directional TOPO Expression kit (CatNo-K4900-40-Invitrogen), and cloned by transforming One-shot TOP 10 Chemically Competent Escherichia coli (Invitrogen), according to the manufacturer's protocol.Plasmids were isolated by using a Plasmid DNA MiniPreps kit (EasySpin), according to manufacturer's instructions.
Sequencing reactions were performed with BigDye ® Terminator v3.1 Cycle Sequencing kit (CatNo-4337456-Applied Biosystems).The automated sequencing was performed in an ABI Prism™ 310 Genetic Analyzer (Applied Biosystems) and analyzed with Sequencing Analysis 3.4.1 software.

Real-time pcr and human metastasis array
Real-time PCR was performed using 1 µl of cDNA, synthesized as described above, and SYBR ® Green Master Mix (CatNo-4309155-Applied Biosystems), according to the manufacturer's instructions.RNA 18S was used as housekeeping gene.Samples were analyzed in triplicate.Reactions were developed in ABI Prism ® 7900HT (Applied Biosystems).
For apoptosis analysis, cells were rinsed with PBS 1X and ressuspended in 0.1% (w/v) BSA in PBS 1X.Cells were incubated with FITC-Annexin V (CatNo-640906-BioLegend) (diluted in annexin binding buffer, 1:40) for 15 minutes, at room temperature.Cells were rinsed with 0.1% (w/v) BSA in PBS 1X and ressuspended in annexin-V binding buffer.Acquisitions were performed using a FACSCalibur (BD Biosciences) and further analyzed with CellQuest Pro software.
Cell aggregation assay was performed in triplicate by coating wells in a 48-well plate with 150 µl of soft-agar solution with subsequent cell seeding (4 × 10 4 cells per well).Aggregation was evaluated at 24 h under an Olympus CK2 inverted optical microscope (Olympus).
For in vitro wound healing assay, cells were grown in a 3.8 cm 2 tissue culture wells to a confluent monolayer.In each well, a scratch was made with a P20 pipette tip to the length of the well.After the scratch, the culture medium was replaced to remove detached cells.A time-lapse experiment was performed and followed under an Olympus CK2 inverted optical microscope (Olympus).Phase images were acquired at the following time points: 0, 8, 24 and 36 hours.

Statistics
Data were analyzed using t-tests (two-tailed) with Excel (Microsoft) and GraphPad prism 5. Statistically significant changes were determined at p < 0.05.

Generation of hct15-fn colorectal cancer cell line
To study the role of FN in modulating tumor onset and progression, a colon adenocarcinoma cell line (HCT15) over expressing FN was generated, by stable transfection with human embryonic FN gene (full length FN), pcDNA3-flFN.This cell group was compared to its wild type counterpart (HCT15 control) in in vitro and in vivo assays.As shown in figure 1A, FN-transfected cells over expressed FN at the mRNA level; this was further confirmed at the protein level (Figure 1B).Interestingly, HCT15-FN cells also over expressed integrins alpha3 and beta 3, when compared to HCT15 control cells.

Fn overexpression results in increased cell proliferation, larger cell aggregates and reduced apoptosis
To evaluate the role of FN over expression in cell proliferation, growth curves were performed, at different time points -0, 3, 6, 24 and 30 hours (Figure 1C).Data showed HCT15-FN had a higher growth rate than HCT15 control cells.
The effect of FN over expression on cell-cell adhesion was also evaluated, by slow aggregation assay (Figure 1D).An increase in cellcell adhesion was observed for cells over expressing FN, as well as the formation of larger aggregates than in HCT15 control cells.
Apoptosis was assessed by Bax/Bcl-2 ratios (Figure 2A) and annexin V staining (Figure 2B).Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) expressions were determined by Western blotting.The results showed a lower Bax/Bcl-2 ratio for HCT15-FN, in comparison to HCT15 control cells, suggesting a lower activation of apoptosis.
Annexin V staining was used to quantify the number of cells undergoing apoptosis within each cell group (Figure 2B).Data showed HCT15-FN cells undergo apoptosis at a statistically significant (p < 0.05) lower rate than HCT15 control cells, at 3 and 30 hours time points.

Zymography
Culture supernatants were concentrated by using Amicon ® Ultra-4 Centrifugal Filter Units (Millipore).SDS loading buffer 5X was added to each sample and the mixtures were loaded in a 12% polyacrilamide gel with 0.1% (w/v) gelatin.Electrophoresis was performed with a Mini-PROTEAN Tetra Electrophoresis System (Bio-Rad), 150 V, for 90 minutes, into TGS buffer 1X (Bio-Rad).Gel was incubated in renaturating buffer (25% TritonX-100 (v/v)) for 1 hour, with agitation, and then gel was incubated, overnight, 37 ⁰C, in collagenase buffer.Staining was performed with 0.5% (w/v) Coomassie Blue R-250 (CatNo-20278-Thermo Scientific) for 30 minutes and destaining with destilled water, with agitation.The gel was scanned and bands were quantified using Image J software.
After 2 months, mice were euthanized in a CO 2 chamber.
Tumor volume was determined as (minor axis 2 × major axis)/2.Tumors and main target organs for metastasis were collected for histological analysis.

Sdf-1:cxcr4 axis blockade reduces tumor progression and metastasis in vivo
Considering the relevance of the SDF-1:CXCR4 axis in cancer progression [22,23], we sought to investigate if this chemokine could be responsible for the overall effects of FN over expression.For this purpose, and after the confirmation of the differential expression of CXCR4, at protein levels (Figure 3C), in vivo experiments were performed to assess the effect of FN over expression on tumor growth and to determine the effect of CXCR4 blockade in the FN-mediated effects.First, the tumorigenic capacity of HCT15 control and HCT15-FN cells was assessed by generating orthotopic xenograft tumors in 6-weeks-old BALB/c-SCID mice.As shown in figure 3A, survival was longer in mice inoculated with HCT15 control, with all animals alive at the end of the experimental period; in contrast, for mice inoculated with HCT15-FN, the overall survival was reduced by 75% at the end of the experiment and mice had to be euthanized.In parallel, we treated mice inoculated with HCT15 control or HCT15-FN cell lines with CXCR4 antagonist-AMD3100.Notably, 100% of mice undergoing AMD3100 treatment survived until the end of the experiment, from HCT15 control and HCT15-FN groups.
Necropsy revealed larger primary tumors in mice inoculated with HCT15 overexpressing FN, when compared to mice inoculated with HCT15 control cells (Figure 4B and Figure 4C).Tumors from mice treated with AMD3100 were however smaller than the respective control group (Figure 4C).Concerning metastasis, histological analysis of liver and lungs revealed the presence of more micrometastases in mice injected with HCT15-FN cells.In sharp contrast, mice treated with AMD3100, regardless of being inoculated with HCT15 control or HCT15-FN cells, did not develop metastases (Figure 4D).

Fn overexpression is associated with higher proteolytic activity and higher migratory capacity of hct15 cells
Another attribute of cancer is the ability of cells to migrate from the original location to invade the surrounding tissues and, ultimately, metastasize at distant organs [21].During these stages, ECM proteolysis is crucial and MMPs assume an important role in this context [5,7].
FN ability to interfere in MMPs production/secretion and/ or activity was assessed by gelatinolytic zymography of cell culture conditioned media (Figure 2C).The results revealed higher enzymatic activity of MMP-9 and MMP-2 in HCT15-FN cells than in HCT15 control cells.
In vitro wound healing assay was performed to assess the role of FN on the migratory rate of cells, by comparing HCT15 control and HCT15-FN (Figure 2D and Figure 2E).HCT15-FN showed a higher migratory capacity than HCT15 control cells.

Fn ovexpression induces cxcr4 and flt-4 expression
We exploited the mechanisms underlying the phenotypes described above, related to FN over expression.For this purpose, an array of genes related to human metastasis was carried out for HCT15 control and HCT15-FN.Data were validated by a second real-time PCR (Figure 3A).Statistically significant (p < 0.05) higher expression of CXCR4, FLT4 and MMP13 and lower expressions of MTA1 and TIMP3 were found for HCT15-FN in comparison to HCT15 control cells.
Given these results, another real-time PCR was carried out targeting CXCR4 and FLT4 ligands -SDF-1 and VEGF C, respectively (Figure 3B).A statistically significant (p < 0.05) increase in SDF-1 and VEGF C expression was also seen in HCT15-FN in comparison to HCT15 control cells.suggested by some studies using different cancer models [14,15,[31][32][33].The correlation between FN and MMPs expression was further supported from data obtained by performing a PCR-metastasis array, which showed increased expressions in MMP-13 and tissue inhibitor of metalloproteinases-3 (TIMP-3), which target MMPs including MMPs -2 , -9 and -13 [7].
As mentioned, ECM proteolysis, mainly by MMPs, is one of its most important events accompanying cell migration [34].Data reported here, showing increased migration and MMP activity by HCT15-FN cells, are therefore in agreement.
Next, we exploited the mechanisms underlying the correlation between FN and further tumor progression, by performing a screening for human metastasis targets.Results revealed an increased expression of CXCR4 and FLT4 and their respective ligands SDF-1 and VEGF C in HCT15-FN in comparison to HCT15 control cells.
Considering the wide spectrum of roles of SDF-1:CXCR4 axis [22,23], we hypothesized this could be an important causal link between increased FN amounts and a more aggressive phenotype in colorectal cancer.
To corroborate the in vitro findings and to validate this assumption, two in vivo experiments were developed in parallel.First, survival of mice inoculated with HCT15-FN was shown to be lower than that of mice injected with parental/control cells.Tumors formed by HCT15-FN cells were also larger than those formed by HCT15 control cells (Figure 4B and Figure 4C); and mice inoculated with HCT15-FN showed more metastases than HCT15 control mice (Figure 4D).These observations are consistent with the in vitro results.Second, CXCR4 blockade was shown to promote survival by reducing tumor growth and metastases formation both in mice inoculated with HCT15 control and, more significantly, in HCT15-FN cells.
Taken together, we assumed that SDF-1:CXCR4 axis emerges as a causal link between increased FN amounts and a more aggressive phenotype in colorectal cancer.A role for SDF-1:CXCR4 has already

Discussion
ECM represents a critical player in oncogenic transformation.The several steps of cancer are largely dependent on the permissive nature of the microenvironment and ECM proteolysis assumes great importance in this context [24][25][26].FN is one of the most abundant components of ECM [2] and several studies related FN levels to tumor progression: in cancer patients, an increase in both FN and FN fragments levels was observed [6,10,11,27] and, in several reports where functional studies on cancer cell lines were performed an equivalent correlation has also been observed [11,[13][14][15][16].
In the context of solid tumor growth, colorectal cancer assumes a relevant position as one of the most common and lethal malignancies worldwide [28].In order to explore the role of FN in tumor development and progression, colon cancer was the chosen model.For this purpose, a stably FN-overexpressing HCT15 cell line was generated, which features were compared to its wild type counterpart (HCT15 control) in in vitro and in vivo assays.
Interestingly, the increase in FN in HCT15-FN cells was accompanied by an increase in α3 and β3 integrins expression, which may suggest a cross-regulatory mechanism, since these integrins are FN receptors [29].
Subsequent detailed characterization of the FN-transfected cell line versus its parental counterpart revealed that the first had a higher proliferative rate, was able to form larger cell aggregates, showed lower levels of apoptosis and increased migratory capacity.MMP-9 and MMP-2 enzymatic activities were also increased in HCT15-FN, accounting for the increased invasion capacity.Besides the evidence that MMPs activity is strongly associated with tumorigenesis [7], MMP-9 and MMP-2 are thought to be crucial for metastases formation [5,8,9,30].These increases in MMPs activity might be related to higher amounts of FN in supernatants of HCT15-FN cells, which is cleaved by MMPs [7,11], and/or FN may activate signaling pathways responsible for MMPs regulation, as it has been been suggested in other cancers, including breast [35], lung [36], prostate [37] and pancreatic cancer [38].CXCR4 expression was also shown to be associated with poor prognosis in colorectal cancer patients [39,40] and with the presence of distant metastases in patients with colorectal cancer, as well as with tumor cells migration in vitro [41,42].Studies on prostate cancer cell lines also linked CXCR4 to increased cell migration and metastasis, through its regulation of MMPs [43,44] and VEGF [43].
On the other hand, the inhibition of either the expression or function of CXCR4 produced a beneficial (clinical) effect in different neoplasms [35,[45][46][47][48][49].Moreover, the SDF-1:CXCR4 axis has been related to ECM components, namely FN, in malignancy contexts.A study on breast cancer showed the involvement of chemokines in tumor cells migration and motility, attributing an important role of CXCR4 in guiding breast cancer cells to target organs, due to a cross-signaling with integrins in an ECM-dependent way [35].SDF-1-induced integrin activation, through CXCR4, in small cell lung cancer, also appeared to co-operate in mediating adhesion, namely to FN, and chemoresistance [49].SDF-1 was also shown to affect the metastasis-related behavior of colorectal cancer cells expressing CXCR4, including an increase in cell proliferation, cell adhesion to FN and cell migration [42].

Conclusions
In conclusion, our findings suggest an active role for FN in promoting colorectal cancer progression and metastases formation, via integrins modulation and by promoting SDF-1:CXCR4 interaction.These studies shed light on the mechanisms whereby an ECM component perturbs and favors neoplastic progression, and reveals novel targets for therapeutic intervention in a setting of invasive colorectal cancer.

Figure 2 :
Figure 2: FN over expression reduces apoptosis and is associated to a higher cell migration rate.A) Assignment of Bax/Bcl-2 ratios, by Western blotting, for control and FN-transfected cells.β-actin was used as housekeeping protein.One hundred µg of protein were loaded in each lane.Bands were quantified using ImageJ software; B) Apoptosis analysis by annexin V -7AAD staining in control and HCT15-FN at 0, 3, 6, 24 and 30 hours ( * p < 0.05); C) Zymography of cells media supernatants.D, Wound healing assay for control and transfected cells, at 0, 8, 24 and 30 hours.Phase microscopy (original magnification: 200x); E) Quantification of wound closure, at 0, 8, 24 and 30 hours.Bands were quantified using ImageJ software.Error bars represent standard deviation.

Figure 3 :
Figure 3: Screening for metastasis-related targets.A) Validation of PCR array for human tumor metastasis by real time PCR.Relative quantification results were compared between control and HCT15-FN ( * p < 0.05); B) Real-time PCR for SDF-1 and VEGF C expression.Relative quantification of mRNA levels in control and FN-transfected cells ( * p < 0.05); C) Immunoreactivity for CXCR4 in control and FN-transfected cells.Fluorescence microscopy (original magnification: 200x).Nuclei were stained with DAPI (blue).Scale bar: 100 µm.Error bars represent standard deviation.

Figure 4 :
Figure 4: Orthotopic xenograft tumors induction of control and HCT15-FN under standard conditions and upon CXCR4 antagonist AMD3100 treatment.A) Kaplan-Meier survival curves for mice inoculated with control and HCT15-FN, over 8 weeks; B) Macroscopic observation of xenograft orthotopic BALB/c-SCID.Representative images of the animals inoculated with control and FN-transfected cells; C) Primary tumors volumes ( * p < 0.05); D) H&E and immunofluorescense (anti-CK19; FITC).Representative images of the lungs of animals inoculated with control and HCT15-FN, not injected and injected with AMD3100, in H&E black arrows indicate metastasis foci.Light and fluorescence microscopy (original magnification: 100x).Scale bar: 50 µm.Error bars represent standard deviation.