Fc receptor-like 1 (FCRL1) is a novel biomarker for prognosis and a possible therapeutic target in diffuse large B-cell lymphoma

doi:10.21203/rs.3.rs-2111137/v1

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Research Article

Fc receptor-like 1 (FCRL1) is a novel biomarker for prognosis and a possible therapeutic target in diffuse large B-cell lymphoma

https://doi.org/10.21203/rs.3.rs-2111137/v1

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Background

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma (NHL), which can involve various types of mature B-cells. The incidence of DLBCL has been increased, additional research is required to identify novel and effective prognostic and therapeutic molecules. Fc receptor-like 1 (FCRL1) act as an activation co-receptor of human B-cells. Aberrant expression of this molecule has been reported in a number of B-cell-related disorders. Moreover, the clinical significance and prognosis value of FCRL1 in DLBCL not completely identified.

Methods

In this study, the expression levels of FCRL1 was determined in thirty patients with DLBCL and 15 healthy controls (HCs). In addition, the correlation between FCRL1 expressions with clinicopathological variables of DLBCL patients were examined. Then, the potential roles of FCRL1 in proliferation, apoptosis, and cell cycle distribution of B-cells from DLBCL patients were determined using flow cytometry analysis, after knockdown of this marker using retroviral short hairpin RNA interference. Quantitative real time-PCR, western blotting, and enzyme-linked immunosorbent assay were also used to identify the possible effects of FCRL1 knockdown on expression levels of BCL-2, BID, BAX, intracellular signaling pathway PI3K/p-Akt, and p65 nuclear factor-kappa B (NF-κB) in B-cells of DLBCL.

Results

Statistical analysis revealed higher levels of FCRL1 expression in B-cells of DLBCL patients compared to HCs at both protein and mRNA levels. A positive correlation observed between the expression of FCRL1 with some clinicopathological parameters of DLBCL patients. In addition, FCRL1 knockdown significantly decreased cell proliferation and stimulated apoptosis as well as G1 cell cycle arrest in B-cells of DLBCL patients. The levels of p65 NF-κB and PI3K/p-Akt expression markedly reduced after knockdown of FCRL1 expression.

Conclusions

These results suggested FCRL1 as a potential novel biomarker for prognosis and/or a possible effective therapeutic target for treatment of patients with DLBCL.

Fc receptor-like 1(FCRL1)

Diffuse large B-cell lymphoma (DLBCL)

Apoptosis

Proliferation

Cell cycle

p65 NF-κB

PI3K/p-Akt

Non-hodgkin's lymphoma (NHL) is a diverse group of cancers that has been classified into 54 subtypes based on the cellular origin. About 90% of NHL types originates from B-cells [1]. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, with a broad spectrum of immunophenotypic, morphological, and clinical features, accounting for approximately 80% of aggressive lymphomas and 30–40% of adult NHL cases [2, 3]. DLBCL can involve various types of mature B-cells and developed in both nodal and extra-nodal sites. The gene expression profiling studies have been introduced three distinct molecular subtypes of DLBCL based on the cell origin and overall survival rates including germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL) [1–4]. The GCB type of DLBCL derived from germinal center (GC) centroblasts and reveal a better diagnosis and survival rate compared to the ABC group. In addition, GCB DLBCLs can be treated with a combination of current therapeutic methods such as chemotherapy and monoclonal antibodies [2].

In recent years, numerous studies have been performed on the pathogenesis and molecular biology of DLBCL disorder to improve the overall survival rates of patients with this type of B-cell hematological malignancy [4–6]. However, the incidence rate of DLBCL has been increased in both genders and all age groups. Approximately 40–60% of patients with DLBCL could be cured and responded to available therapeutic methods [2, 3]. Therefore, additional researches are required to identify novel prognostics and effective therapeutic molecules, which might improve prognosis, treatment, and survival rates of patients with DLBCL.

Fc receptor like (FCRL) proteins (also known as FcRH, IRTA, IFGP, SPAP, and BXMAS) belongs a subgroup of lymphocyte receptors with immunoregulatory potential that preferentially expressed by B lineage cells. FCRL genes are located on human chromosome 1q21-23 and encode two cytoplasmic proteins FCRL-A, B and six transmembrane glycoproteins FCRL1-6 with varying numbers of Ig-like domains on their extracellular regions and immunoreceptor tyrosine-based activating/ inhibitory motifs (ITAM/ ITIM) in their cytoplasmic domain [7–9]. The structural properties and expression profile of FCRL molecules have proposed the regulatory potential of these receptors on human B-cell responses [10–12]. Moreover, several groups of researchers have revealed a clinical relevance between the aberrant expression of these proteins with different disorders such as autoimmune diseases [13–15], infections [16, 17], and various types of B-cell related hematological malignancies [18–24]. Among these receptors, FCRL1 is a pan B-cell marker with two ITAM-like sequences in cytoplasmic domain and glutamic amino acids with negative charges in the transmembrane region. This receptor differentially expressed during B-cell development [25]. FCRL1 has been introduced as an activation co-receptor with a great potential to improve the function of human B-cells [26]. The aberrant expression of this molecule was reported in several NHL cell lines and many B-cell related leukemia and lymphoma disorders including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and hairy cell leukemia (HCL) [21, 22, 24]. However, there is very little research on the clinical significance and prognostic value of FCRL1 in DLBCL.

This study was performed to evaluate the levels of FCRL1 expression in diagnosed patients with DLBCL compared to healthy controls (HCs). The correlation between the expression levels of FCRL1 with clinicopathological features of these patients were determined. In addition, we were investigated the potential effects of FCRL1 in development of DLBCL through evaluation of apoptotic cell death, proliferation, cell cycle distribution, and expression levels of important nuclear transcription factor (NF)-κB and intracellular signaling pathway phosphoinositide 3-Kinase (PI3K) /phosphorylated (p)-Akt in B-cells of DLBCL patients, following knockdown of FCRL1 expression.

Cell culture

The Platinum-A (Plat-A) cell line (Cell Biolabs, Inc., USA), a packaging retroviral cell line, was cultured in Dulbecco^’s Modified Eagle^’s Medium (DMEM)-high glucose medium (Gibco, USA) with 10% FBS (Gibco), 2 mM L-glutamine (Gibco), 1% penicillin-streptomycin (ICN Biomedicals, OH), 1x non-essential amino acids (Gibco), and 1% blasticidin S (Life Technologies, NY). The cells were maintained in a CO₂ incubator with 95% humidified air at 37 ^οC.

Subjects

In this research, according to previous studies and using StataCorp. 2003. Stata Statistical Software: Release 8. College Station, TX: StataCorp LP, the minimum sample size was determined to be 28 and 14 for patients and HCs groups, respectively. In total, 30 newly diagnosed patients with DLBCL were included in the study. Two third of the patients were male and 10 patients were female (mean age 63.7 ± 15.5). According to the immunohistochemistry (IHC) results and their medical records, 10 out of 30 patients (33.3%) had GCB-DLBCL and other patients (66.7%) showed non-GCB subtypes of DLBCL. The clinical characteristics of these patients are listed in Table 2. In DLBCL patients, the biopsy specimens were collected from different sources, including 24 lymph node cases, 2 soft tissue cases, 2 tonsil cases, 1 spleen case, and 1 gastrointestinal tract case. The specimens is placed into a sterile conical tube containing DMEM as the transport medium. In addition, 15 cases with reactive hyperplastic lymph nodes without any familial history of various malignancies were included in the study as HCs including 9 males and 6 females (mean age of 59.3 ± 16.8).

Table 1

Primers used in quantitative real-time reverse transcriptase-polymerase chain reaction.
Gene	Forward/ reverse	Sequence
FCRL1	Forward	5'-CAGAGTTCAGATGCCCAGTT-3^'
	Revers	5'-TCACATCAGCAGGGAC-3^'
BCL2	Forward	5'-GGGATGCGGGAGATGTGG-3^'
	Revers	5'-GTAGCGGCGGGAGAAGTC-3^'
BID	Forward	5'-CATCCGGAATATTGCCAGGC-3^'
	Revers	5'-CCATGTCTCTAGGGTAGGCC-3^'
BAX	Forward	5'-AACAAGCTGAGCGAGTGTCT-3^'
	Revers	5'-GTTCTGATCAGTTCCGGCAC-3^'
PI3K	Forward	5'-CTTCCTCCACCTCTTTGCCCTG-3^'
	Revers	5'-AGCCACTACTGCCTGTTGTCTTG-3^'
β-actin	Forward	5'-GGACTTCGAGCAAGAGATGG-3^'
	Revers	5'-AGCACTGTGTTGGCGTACAG-3^'

Table 2: Association between different levels of FCRL1 protein expression with clinicopathological features of newly diagnosed of patients with DLBCL.

		FCRL1 protein expression (MFI)			Variable n
*^P-value*	χ2	High (n=16; 53.3%)	Intermediate (n=7; 23.3%)	Low (n=7; 23.3%)

0.004 0.81						Age (years)
		0	1	2	3	< 40
		6	2	3	11	40-60
		10	4	2	16	> 60
2.2 0.53						Gender
		11	5	4	20	Male
		5	2	3	10	Female
5.98 0.02						Tumor Size (cm)
		7	3	5	15	<4
		9	4	2	15	≥4
6.31 0.015						Subtype
		3	3	4	10	GCB
		13	4	3	20	Non-GCB
7.81 0.007						Stage
		7	3	5	15	Stage I-II
		9	4	2	15	Stage III-IV
2.9 0.23						Extra-nodal status
		10	4	5	19	0-1
		6	3	2	11	>1
4.91 0.07						LDH (U/L)
		7	4	6	17	Normal
		9	3	1	13	High
8.52 0.003						IPI score
		12	3	5	20	0-2
		4	4	2	10	≥3

DLBCL diffuse large B-cell lymphoma, n number, MFI mean fluorescent intensity, GCB germinal center B, LDH lactate dehydrogenase, IPI international prognostic index.

Cd19 + b Cells Isolation

The tissue disaggregation method and magnetic-activated cell sorting (MACS) approach was performed to prepare human B-cells from tissue samples, as previously mentioned [27, 28]. Briefly, each tissue sample was transferred into a sterile petri dish (Nunc- Nalgene, USA), cut into 2–3 mm diameter pieces, and incubated for 60 minutes at 37 ^οC with a combination of serum-free DMEM-high glucose medium (Gibco), collagenase (Sigma-Aldrich, Germany), trypsin (Gibco), and DNAse (Sigma-Aldrich). Then, residual activity of trypsin was neutralized and single-cell suspensions prepared by injection of complete DMEM-high glucose medium (Gibco) into the solid tissues with a pushing and pulsing action, until cells are released. The cell suspensions were collected and centrifuged for 5 minutes at 1300 rpm. To remove the red blood cells (RBCs), the cell pellet is resuspended in 5 ml ammonium chloride solution and incubated for 5 minutes at room temperature. Human CD19 + B-cells were isolated from cell suspensions using CD19 positive selection kit and AutoMACS Pro Separator (Miltenyi Biotech, USA) according to the kit's instructions. The cell counts and percentage of cell viability were elucidate using a hemocytometer and trypan blue dye exclusion method. In addition, purity of isolated B-cells was determine using flowcytometry assay and fluorescein isothiocyanate (FITC)-labled anti-human CD19 antibody. In each sample, cell viability and purity ≥ 90% was acceptable for further investigation. Then, isolated B-cells were cultured in DMEM-high glucose medium supplemented with 1% penicillin- streptomycin, 2 mM L-glutamine, 1x non-essential amino acids, 15% FBS (all from Gibco), and 0.25 M B-class CpG oligodeoxyribonucleotide (CpG-ODN) (Miltenyi Biotech) for 5–7 days, until B-cell aggregation and increase in the number of cells were visible.

Evaluation Of Fcrl1 Protein Expression

The flowcytometry assay was used to evaluate the levels of FCRL1 protein expression in various cell lines and prepared CD19 + B-cells. To determine the expression levels of FCRL1 in malignant B-cells of diagnosed patients with DLBCL compared to HCs, cultured CD19 + B-cells were stained with a combination of Phycoerythrin (PE) anti-human FCRL1 (Miltenyi Biotech; clone: REA440), Pacific Blue anti-human CD19 (Biolegend, USA; clone: HIB19), PerCP-Cy5.5 anti-human CD20 (BD Biosciences, USA; clone: L27), Allophycocyanin (APC) anti-human/ mouse BCL6 (Miltenyi Biotech; clone: REA373), and Alex Flour® 647-labeled anti-BCL2 (Biolegend; clone: 100) antibodies or matched isotype control antibodies. In addition, the fluorescence minus one (FMO) controls were used to confirm the correct cell population gating and fluorescence spread. In patient’s samples, CD19 + CD20 + BCL2 + BCL6 + cells were selected as the malignant B-cells to analysis the expression levels of FCRL1. In addition, malignant cells were examined for dual kappa/lambda light-chain expression. Data were analyzed by a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA), and assessed using FlowJo software (v10.1, FlowJo, Ashland, OR, USA).

Preparation Of Retrovirus Packaging

To determine the potential roles of FCRL1 on DLBCL development, we selected retrovirus expression system for knockdown of this molecule in B-cells of patients with DLBCL. A high levels and more efficient of FCRL1- retrovirus is essential for efficient transduction of human B-cells in vitro. In order to, a potent packaging retroviral cell line Plat-A and retroviral GFP vectors (OriGene Technologies, USA, Locus ID 115350) were used in this experiment. This vector containing four unique 29-mer shRNA constructs against different splice variants of the FCRL1 genes. Also, non-targeting scrambled shRNA in pGFP-V-RS vector used as negative control to assess the efficiency of FCRL1 knockdown process. The shRNA constructs was transformed in Escherichia coli strain DH5α and selected on antibiotic plates to detect resistance plasmids [29]. The antibiotic-resistance bacteria were grown overnight in LB-agar media and then accuPrep Plasmid Maxi-Prep DNA Extraction Kit (Bioneer, Korea) was used to extract the E.coli DH5α plasmids, according to the kit instruction.

The calcium phosphate (CaPO4) precipitation method was performed to transient transfection of Plat-A cells and production of retroviral particles, due to easy and safety of this method [30]. The transfection efficiency was determined based on the GFP signals under the fluorescence microscopy. The supernatant of Plat-A cells containing retroviral particles were collected at 48h and 72h post-transfection, filtered with 0.45µm syringe filters (Millipore, USA) to remove the cell debris, and stored at -80°C until used for viral transduction in human B-cells.

Retroviral Transduction

Spin infection is the most efficient method for viral transduction of suspended human blood cells such as B-cells. Transduction duration, use of 8 µg/ml polybrene, and suitable density of the target cells are crucial factors to increase the efficiency of this protocol. The wells of 24 well cell culture plates seed with a combination of 1 ml retrovirus solution, 10 µg/ml goat F(ab')2 anti-human IgG/IgM (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), 8 µg/ml polybrene (Santa Cruz Biotechnology, Dallas, Tx), 0.25 µM B-class CpG-ODN (Miltenyi Biotech), and 3 × 10⁶ prepared B-cells of DLBCL patients. The plates were centrifuged at 2500 rpm for 90 minutes, and maintained in a CO₂ incubator with 95% humidity and 37 ^οC temperature. The efficiency of transduction and knockdown of FCRL1 expression were determined using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting after 48 and 72h of incubation.

In continue, we indicated the treated cells and control cells phrases to describe the isolated B-cells that transduced with retroviral particles harboring FCRL1-targeting DNA or control vector DNA, respectively.

Quantitative Real- Time Pcr

To evaluate the genes expression levels, total RNAs were extracted from 1 to 1.5 × 10⁶ target cells by RNAx plus solution (Cinagen Company, Iran) according to the manufacturer’s protocols. The quality and concentration of extracted RNAs were determined using the NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). The same concentrations of extracted RNAs reverse transcribed to first-strand complementary deoxyribonucleic acid (cDNA) using random hexamer and one-step SYBR PrimeScript RT Reagent Kit (Takara, Japan) according to the manufacturer's guidelines. The qRT-PCR was performed on a rotor-gene 6000 instrument (Qiagen, Germany) in a 20µl reaction mixture for each sample containing 2µl of template cDNA, 10µl of SYBR Green PCR Master Mix (Takara), and appropriate amounts of each primer and DNase-RNase free water. Each reaction was incubated at 95°C for 30 sec, followed by 45 cycles (FCRL1), 30 cycles (BCL2, BID), and 35 cycles (BAX, PI3K) of 95 ^οC for 5 sec and 60 ^οC (FCRL1, BCL2 and BID) or 61^οC (PI3K and BAX) for 30 sec. The Primers were designed using AlleleID 7.0 software (Premier Biosoft, USA). Sequences of primers are shown in Table 1. Human β-actin was used as an endogenous control to normalize the expression level of each gene. Relative expression levels of the target genes were calculated using the Relative Expression Software Tool 2009 (REST 2009) and reported as 2^−ΔΔCt [31, 32].

Western Blotting

Western blotting was performed to detect the expression levels of FCRL1 and phosphorylated Akt (p-Akt) proteins in prepared B-cells of DLBCL patients. The cells were collected and washed three times with phosphate-buffered saline (PBS, pH 7.2), 72 h post retrovirus transduction. Target cells were lysed in RIPA buffer with protease and phosphatase inhibitors and protein concentration was determined using the bicinchoninic acid (BCA) kit (Sigma-Aldrich, Germany). The protein samples (40µg per sample) were separated by 12–15% SDS-PAGE gel, transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore), and blocked with 5% skimmed milk in Tris Buffered Saline with Tween (TBST) at room temperature for 2 h. The membranes were incubated with primary antibodies against FCRL1 (0.5µg per ml, catalog number MAB20491, R&D Systems, USA), p-Akt (S473) (0.2µg per ml, catalog number MAB887, R&D Systems), and β-actin (1:4000, catalog number 66009-1-LG, Proteintech) at 4°C overnight with gentle shaking. They were washed three times with TBST and incubated with a horseradish-peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Finally, the expression levels of target proteins were assessed using enhanced chemiluminescence (ECL) assay.

Detection Of Intracellular Proteins By Flowcytometry

The expression levels of intracellular proteins p-Akt (S473), BCL2, and BCL6 were determined by flowcytometry analysis, according to the established protocols. Briefly, target cells were fixed with 0.01% formaldehyde for 20 minutes at room temperature and permeabilized with Tween 20 (0.1% v/v in PBS, pH 7.2) for 15 minutes in the dark at room temperature. Then, intracellular staining of cells was performed with a combination of Alex Flour® 647-labeled anti-BCL2 (Biolegend; clone: 100), APC anti-human/ mouse BCL6 (Miltenyi Biotech; clone: REA373), and fluorescein isothiocyanate (FITC)-labeled anti-human p-Akt (S473) (eBioscience; clone: CA) antibodies or matched isotype control antibodies for 30–45 minutes at 4 ºC. The expression of these proteins were also determined by FACSCalibur flow cytometry (BD Biosciences) and then analyzed using FlowJo software.

Cell Proliferation

The effects of FCRL1 knockdown on the rate of B-cell division was evaluated using cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (Biolegend) according to the manufacturer’s instructions. In brief, cultured B-cells of DLBCL patients (1 × 10⁶) were re-suspended in PBS and incubated with 0.5µM CFSE (Biolegend) for 8 minutes in the dark at room temperature, followed by the extensive washing of cells with complete DMEM high-glucose medium. The CFSE-labeled B-cells were transduced with FCRL1-retroviral particles based on the protocol, which has previously described in the “transduction of B-cells” section, and incubated in a CO₂ incubator with 37 ^οC temperature. The cells were harvested at days 3 and 5 post-transduction and washed three times in PBS. The percentage of B-cells proliferation were determined using FACSCalibur flow cytometry (BD Biosciences) and assessed by FlowJo software based on the fluorescent intensity of CFSE dye. The non-labeled B-cells were used to exclude cell auto-fluorescent.

Apoptotic Cell Death And Cell Cycle Analysis

The potential roles of FCRL1 on B-cells apoptosis and cell cycle distribution were examined using flowcytometry analysis. The percentage of apoptotic cell death were evaluated using the FITC Annexin V apoptosis detection kit (BD bioscience). The cultured B-cells were collected, and washed twice in PBS, 48 and 72 h after retroviral transduction. The cells re-suspended with a solution for FITC Annexin V binding and stained with 5µl Annexin V FITC and 5µl propidium iodide (PI). Samples were incubated in the dark for 15 minutes at 4ºC, analyzed using a BD FACSCalibur flow cytometry, and assessed using FlowJo software.

To determine cells cycle phases, B-cells were collected 72 h post-transduction, fixed in ice-cold 70% ethanol overnight, and incubated with 50 µg/ml PI (Sigma-Aldrich), 0.1% Triton X-100, and 10 µg/ml DNAse-free RNaseA (Sigma Aldrich) in the dark for 15 minutes at 37 ºC. Cell cycle progression were assessed by FlowJo software.

P65 Nf-κb Detection By Enzyme-linked Immunoabsorbent Assay

The expression levels of p65 NF-κB was measured by NF-kB p65 (Total) Multispecies InstantOne Enzyme-linked immunoabsorbent assay (ELISA) Kit, (eBioscience) according to its protocol. Briefly, the B-cells were collected 72 h after transduction procedure, washed twice in PBS, resuspended in 1 ml DMEM-high glucose medium containing 0.5% FBS, and incubated at 37 ^οC for 30 minutes with or without 20 ng/ml of tumor necrosis factor-α (TNF-α) (Miltenyi Biotech). Absorbance was detected at 450 nm with ELISA reader and concentration of this nuclear transcription factor measured based on standard curve and adjusted by dilution factor. Each assay was performed in the duplicate wells and repeated three times independently.

Statistical analysis

Data analysis was performed using IBM SPSS 20 and Microsoft Excel 2016. Bonferroni adjustment and Dunn’s post hoc tests were carried out on each pair of groups. The unpaired t-test and Mann-Whitney U test were used to determine the source of significant variations between two groups with normal and non-normal distribution, respectively. The Fisher’s exact test was also used to identify the correlation between FCRL1 expression and clinical features of patients. Data are presented as mean ± standard deviation (SD), and a p value ≤ 0.05 is statistically significant.

Expression levels of FCRL1

Data showed a significant differences between the expression of FCRL1 in malignant B-cells of patients with DLBCL (CD19⁺ CD20⁺ BCL2⁺ BCL6⁺) compared to B-cells of HCs (CD19⁺ CD20⁺) at protein (p < 0.0001) and mRNA (p < 0.001) levels (Fig. 1A and B).

An example of gating strategy and flow cytometry analysis of FCRL1 protein expression in B-cells of one healthy individual and one patients with DLBCL have shown in supplementary Fig. 1A and B. An initial survey of mean fluorescent intensity (MFI) revealed that DLBCL cases expressed FCRL1 protein with variable range of MFI values, from low levels (MFI = 68.6) to high levels (MFI = 215.18). Therefore, based on the MFI values, we were divided DLBCL patients in three groups with low, intermediate, and high levels of FCRL1 expression. Data analysis demonstrated higher levels of FCRL1 protein expression in 30% (3/10) of patients with GCB-DLBCL subtype and 65% (13/20) of non-GCB DLBCL patients (Table 2).

A positive correlation was found between the expression levels of FCRL1 with multiple clinicopathological features of newly diagnosed patients with DLBCL including tumor size (p < 0.05), stage of disease (p < 0.01), performance status at diagnosis (p < 0.05), and IPI scores (p < 0.01). However, no significant differences was observed between the expression levels of FCRL1with age, gender, lactate dehydrogenase (LDL) enzyme, and extra nodal status of patients (Table 2).

Knockdown Of Fcrl1 Expression

Data analysis revealed significantly decrease in the levels of FCRL1 expression in treated B-cells compared to control and uninfected cells at both mRNA and protein levels (p < 0.01, Fig. 2A and B).

Effects Of Fcrl1 Knockdown On Proliferation, Apoptosis, And Cell Cycle Progression Of Dlbcl B-cells

Inhibition of B-cells proliferation identified in treated B-cells compared to control and uninfected cells, after 3 and 5 days of retroviral transduction procedure and knockdown of FCRL1 expression (p < 0.001- p < 0.05, Fig. 3A and B).

The FCRL1 knockdown were also reduced the expression levels of anti-apoptotic BCL2 gene in treated B-cells than control and/or uninfected B-cells (p < 0.001, Fig. 4A), while the expression levels of pro-apoptotic BID and BAX genes were increased in the treated B-cells (p < 0.01- p < 0.05, Fig. 4A).

Double staining of the cells with FITC Annexin V-PI and flow cytometry analysis showed significantly increase in the percentage of cell apoptosis in treated B-cells compared to control and uninfected cells, 48 and 72h after transduction process and knockdown of FCRL1 expression (p < 0.05) (Fig. 4B and C).

The cell cycle assay was revealed a statistically significant reduction in the percentage of treated B-cells in the S and G2/M phases, after 72 h of transduction procedure. Although, the percentage of treated cells in the sub-G1 (apoptotic cells) and G1 phases were increased than control and uninfected cells (p < 0.01- p < 0.05, Fig. 5).

Effects Of Fcrl1 Knockdown On P65 Nf-κb And Pi3k/p-akt Expression

The results revealed that knockdown of FCRL1 expression significantly decreased the expression levels of phosphorylated-p65 NF-κB in treated B-cells compared with control and uninfected cells in the presence of TNF-α, after 72 h of transduction procedure (p < 0.05, Fig. 6A). The similar results were found in the levels of total p65 NF-κB expression in absence of TNF-α, whether reduction the levels of this protein was not statistically significant (p = 0.072, Fig. 6A).

In addition, a significant decrease in the expression levels of PI3K mRNA and p-Akt (S473) protein were found in treated compared to control and uninfected B-cells (p < 0.01- p < 0.05, Fig. 6B-E ).

DLBCL is an aggressive form of B-cell lymphoma with various pathogenesis, prognosis, and clinicopathological features. Approximately two-thirds of diagnosed patients with DLBCL can be treated with current therapeutic approaches [2, 3]. In addition, most patients diagnosed in the late stages of disease which could increase the rate of mortality. Therefore, identification of unique biomarkers that are associated with pathogenesis and development of DLBCL is essential for early diagnosis and effective treatment of patients with DLBCL. Recently, aberrant expression and potential roles of FCRL1 in the pathogenesis of multiple types of hematological malignancies including HCL, CLL, MCL, and FL have been reported by various studies [11, 18, 19, 21]. In addition, our recent study was revealed higher levels of FCRL1 expression in Iranian patients with DLBCL, HCL, and Burkitt’s lymphoma (BL) compared to HCs [24]. In another study, we also showed the regulatory potential of FCRL1 in BL cell lines Ramos and Daudi [12]. However, there are a limited numbers of studies on potential effects of FCRL1 in DLBCL development. In this study, the expression levels of FCRL1 were evaluated in HCs and malignant B-cells of patients newly diagnosed with DLBCL. In addition, the possible association between the expression levels of this marker with various clinicopathological properties of DLBCL patients were determined. Similar to previous studies, overexpression of FCRL1 was found in malignant B-cells of DLBCL patients when compared with HCs B-cells. Our study additionally showed higher levels of FCRL1 expression in patients with non-GCB subtype of DLBCL compared with GCB-DLBCL patients. The aberrant overexpression of FCRL1 in various subtypes of DLBCL patients may be related to more aggressive status of non-GCB subtype of DLBCL or could link to different expression pattern of FCRL1 during B-cells development. Expression of FCRL1 started in pre B-cells and changed during B-cells development. Higher levels of FCRL1 was detected in immature, mature, and memory B- cells. Although, plasma cells and germinal center B-cells expressed this molecule in very low levels [21, 22, 33]. The results also revealed a positive relationship between FCRL1 expression levels with tumor size, stage, and IPI scores of patients. These findings suggested FCRL1 as an important molecule in the pathogenesis and development of DLBCL as well as expression profile of FCRL1 might serve as a suitable marker for DLBCL prognosis. Then, the biological activities of FCRL1 in DLBCL were investigated. According to the results obtained from a study, FCRL1 has the potential to amplify the B-cell receptor (BCR)-mediated proliferation [26]. As expected, our findings affirmed that knockdown of FCRL1 expression suppressed proliferation of B-cells from DLBCL. This study also showed that FCRL1 knockdown with changing the expression levels of important apoptosis-related molecules including BCL2, BID, and BAX as well as stimulation of G1 cell cycle arrest induced B-cells apoptosis in DLBCL. Moreover, these results were inconsistent with the findings of a related study that was revealed FCRL1 down-regulation had no effect on the expression of different anti-apoptotic and pro-apoptotic proteins [26]. This discrepancy in the potential effects of FCRL1 on B-cell apoptosis might be associated with the differences in the type and sensitivity of applied methods. Therefore, our results showed that FCRL1 might be involved in the pathogenesis or development of DLBCL by regulating of various biological activities of B-cells such as proliferation, cell cycle, and apoptotic cell death.

However, additional investigation is require to determine the underlying molecular mechanisms of activation co-receptor FCRL1 in human B-cells. Different oncogenic pathways involved in the pathogenesis and development of various subtypes of DLBCL. The PI3K/p-Akt and transcription factor NF-κB have been reported as hallmarks of DLBCL biology. The deregulation of PI3K/p-Akt signaling pathway and constitutive activation of key transcription factor NF-κB play critical roles in the pathogenesis of DLBCL through inducing of B-cell survival and inhibition of cell apoptosis [34–36]. PI3K with activation and phosphorylation of serine/threonine kinase Akt at serine 473 (S473) induced TNF-α mediated NF-κB activation, through degradation of IκB inhibitor and phosphorylation of p65 subunit of NF-κB using p-Akt (S473) [34, 35, 37, 38]. The cross talk between key transcription factor NF-κB and PI3K/p-Akt pathway by activation of several cell survival mediators and apoptosis regulating genes in B-cells such as BCL2 genes family, may involve in DLBCL progression [39]. In addition, recent studies in DLBCL indicate activation of NF-κB as a critical mechanism of chemotherapy resistance in ABC-DLBCL as well as suggested NF-κB pathway as an effective target for new therapeutic strategies [38, 40, 41]. A worse clinical outcome and decrease overall survival rate have been reported in DLBCL patients with higher expression of p-Akt [40, 41].

Regarding the potential effects of FCRL1, p65 NF-κB, and PI3K/p-Akt in DLBCL pathogenesis, the possible association between the levels of FCRL1 expression with these oncogenic factors evaluated in B-cells of DLBCL tissues, after FCRL1 knockdown. Our results showed that FCRL1 knockdown significantly reduced the levels of p-p65 NF-κB, PI3K, and p-Akt (S473) in treated B-cells.

This study has some limitations: The small numbers of samples could indicate as important limitation of presented study. DLBCL patients who were included in this study newly diagnosed with DLBCL. The results of the presented study could be more reliable if treated DLBCL patients have been used for examination of FCRL1 expression pattern. In addition, evaluation the possible association between the expression patterns of FCRL1 with other oncogenic molecules involved in the pathophysiology of DLBCL would improve using of FCRL1 as a beneficial target for management of FCRL1 positive B-cell leukemia or lymphoma including DLBCL, which should be investigated in future research.

It might be concluded from the results of the presented study that higher expression levels of FCRL1 in malignant B-cells of DLBCL patients compared to HCs are associated to pathophysiology of this disorder. In addition, the potential effects of FCRL1 knockdown on various activities of B-cells from DLBCL were evaluated and surprisingly, the proliferation of B-cells and expression levels of anti-apoptotic BCL-2 were decreased but apoptotic cell death, cell cycle arrest, and expression of all candidate molecules including pro-apoptotic genes BID and BAX as well as PI3K/ p-Akt and NF-κB were decreased. These results suggested that FCRL1 as an activation co-receptor of human B-cells can be used as a new target for early prognosis and immunotherapeutic intervention of patients with DLBCL, but more investigation with larger sample sizes is required.

Compliance with ethical standards:

Funding

This study was funded by Isfahan University of Medical Sciences (grant number: 394608).

Ethical approval

All procedures performed in this study were in accordance with the ethical standards of the Isfahan University of Medical Sciences committee (ID: IR.MUI.REC.1394.3.608) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent:

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that there is no conflict of interests.

Acknowledgments

The present study was supported by Isfahan University of medical sciences as a PhD Thesis.

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SupplementaryFigure1.jpg
Supplementary Fig. 1. Gating strategy and flowcytometric analysis of FCRL1 protein expression (A) in B-cells of one Healthy individual (B) and malignant/ lymphoma B-cells of one patients with DLBCL.

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Editorial decision: Minor Revisions Needed
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You are reading this latest preprint version

Fc receptor-like 1 (FCRL1) is a novel biomarker for prognosis and a possible therapeutic target in diffuse large B-cell lymphoma

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Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Cell culture

Subjects

Cd19 + b Cells Isolation

Evaluation Of Fcrl1 Protein Expression

Preparation Of Retrovirus Packaging

Retroviral Transduction

Quantitative Real- Time Pcr

Western Blotting

Detection Of Intracellular Proteins By Flowcytometry

Cell Proliferation

Apoptotic Cell Death And Cell Cycle Analysis

P65 Nf-κb Detection By Enzyme-linked Immunoabsorbent Assay

Statistical analysis

Results

Expression levels of FCRL1

Knockdown Of Fcrl1 Expression

Effects Of Fcrl1 Knockdown On Proliferation, Apoptosis, And Cell Cycle Progression Of Dlbcl B-cells

Effects Of Fcrl1 Knockdown On P65 Nf-κb And Pi3k/p-akt Expression

Discussion

Conclusions

Declarations

References

Supplementary Files

Status:

Version 1