Leukemia & Lymphoma
Zi-Zhen Xu, Jian-Kang Shen, Shu-Qing Zhao & Jun-Min Li
ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Clinical significance of chemokine receptor CXCR4 and mammalian target of rapamycin (mTOR) expression in patients with diffuse large B-cell lymphoma
To cite this article: Zi-Zhen Xu, Jian-Kang Shen, Shu-Qing Zhao & Jun-Min Li (2017): Clinical significance of chemokine receptor CXCR4 and mammalian target of rapamycin (mTOR) expression in patients with diffuse large B-cell lymphoma, Leukemia & Lymphoma, DOI: 10.1080/10428194.2017.1379077
To link to this article: http://dx.doi.org/10.1080/10428194.2017.1379077
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Date: 29 September 2017, At: 06:43
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https://doi.org/10.1080/10428194.2017.1379077
ORIGINAL ARTICLE: RESEARCH Image
Clinical significance of chemokine receptor CXCR4 and mammalian target of rapamycin (mTOR) expression in patients with diffuse large B-cell lymphoma
Zi-Zhen Xuaω, Jian-Kang Shenbω, Shu-Qing Zhaoc and Jun-Min Lid
aDepartment of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; bDepartment of Surgery, Luwan Branch of Ruijin Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; cDepartment of Hematology, Luwan Branch of Ruijin Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; dDepartment of Hematology, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China
ABSTRACt
To assess the relevance of C-X-C chemokine receptor type 4 (CXCR4) and mammalian target of rapamycin (mTOR) to large-B-cell lymphoma (DLBCL), levels of protein expression were measured in 56 DLBCL patients who had received rituximab-based therapy. Of these, 34 were positive for CXCR4 expression (60.7%) and 31 for mTOR (55.4%). CXCR4 expression was positively correlated
high lactate dehydrogenase (LDH) level (p ¼ .009), high IPI score (p ¼ .030) and non-GCB subtypewith mTOR expression (r ¼ 0.602; p ¼ .000). CXCR4 expression was significantly associated withwith the chance of remission (p < .05). Kaplan–Meier analysis indicated significantly shorter pro- gression-free survival (PFS) and overall survival (OS) in patients positive for CXCR4 and mTORexpression.
The combination therapy with CXCR4 inhibitor WZ811 and mTOR inhibitor everoli- mus showed syncergistic effect in DLBCL cell lines. These results suggest that the expression of CXCR4 and mTOR may be suitable as biomarkers of the prognosis of DLBCL and for develop- ment of new therapeutic strategies.
(p ¼ .006). Furthermore, the expression levels of CXCR4 and mTOR were negatively correlated
ARTICLE HISTORY
Received 27 February 2017
Accepted 6 September 2017
KEYWORDS
C-X-C chemokine receptor type 4; mTOR; diffuse large B cell lymphoma; rituximab; immunohistochemistry; survival
Introduction
Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin’s lymphoma (NHL) found in adults. It accounts for 30–40% of the total incidence of NHL [1]. DLBCL typically presents as a nodal or extra- nodal mass with rapid tumor growth. Extranodal DLBCL (primary sites are outside the lymphatic system) accounts for 30–40% of DLBCL. Approximately 70% of DLBCL involves at least one and 30% have multiple extranodal involvements [2–3]. Although many patients have shown longer overall survival if treated routinely, with rituximab (an anti-CD20 monoclonal antibody), one-third of patients have a disease that is either refractory to initial therapy or relapses after standard therapy [4]. In view of the genetic heterogen- eity of DLBCL, biological markers associated with cell signaling and trafficking should be identified.
The alpha-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) is specific to stromal-derived- factor-1 (SDF-1, also CXCL12) [5]. CXCR4 plays an important role in the survival, migration, proliferation and metastasis of several cancers including hemato- poietic cancer [6]. CXCR4 and its ligand have been found to be involved in the migration and trafficking of malignant B cells in B-cell non-Hodgkin’s lymphoma [7], lymphoplasmacytic lymphoma [8], mantle cell lymphoma [9] and chronic lymphocytic leukemia [10]. In addition, CXCR4 is considered a metastatic index and a predictor of poor prognosis in DLBCL. It is corre- lated with disease aggressiveness [11–13].
The SDF-1a/CXCR4 interaction has been reported to activate several downstream signaling pathways, such as the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mammalian target of rapamycin (mTOR) pathways [10]. The PI3K/AKT/mTOR signaling is import- ant to the control of proliferation and survival of tumor cells in several types of malignancies, including DLBCL. For this reason, it may be suitable for thera- peutic intervention [14]. Previous studies have con- firmed that abnormal activation of the PI3K-/Akt-/ mTOR-signaling pathway is associated with the development of DLBCL, and it is an indicator of poor prognosis of DLBCL [15]. Because of the importance ofDownloaded by [Australian Catholic University] at 06:43 29 September 2017CONTACT Zi-Zhen Xu Image [email protected] ImageDepartment of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine,
Shanghai, 200025, China
ωThese authors contributed equally to this work.
© 2017 Informa UK Limited, trading as Taylor & Francis Group
Image2 Z.-Z. XU ET AL.
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CXCR4 and PI3K/AKT/mTOR pathway to lymphoma microenvironment cross talk, the expression of CXCR4 and mTOR in DLBCL was investigated using immuno- histochemical methods, and the clinical and patho- logical significance of CXCR4 and mTOR and their predictive value in the prognosis of DLBCL were ana- lyzed. The antiproliferative efficacy of the CXCR4 inhibitor WZ811 alone or combined with the mTOR inhibitor everolimus was further evaluated in DLBCL cell lines. Our results showed that the expression of CXCR4 and mTOR may be suitable as biomarkers of the prognosis of DLBCL and for development of new therapeutic strategies.
Methods
Patients and treatment
This retrospective study was collected data from 56 newly diagnosed DLBCL patients, who received stand- ardized R-CHOP therapy at Ruijin Hospital and Luwan Branch of Ruijin Hospital from January 2006 to June 2010. The diagnosis of DLBCL was performed in accordance with the World Health Organization’s clas- sification system [16]. All selected patients signed informed consent a required by the Shanghai Jiao Tong University School of Medicine ethics committee.
Cells and cell culture
The germinal center B-like (GCB) DLBCL cell lines SUDHL-4(CRL-2957) was obtained from the American Type Culture Collection (Bethesda, MD). The cells were cultured in RPMI-1640 medium (Gibco/BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (PAA, Linz, Austria) in a 5% CO2 – 95% air-
humidified atmosphere at 37 ◦C. The activated B-cell-
like (ABC) DLBCL cell line Ly-3 was a gift from Dr. B. H. Ye (Department of Cell Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY). The cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, Grand Island, NY) supplemented with 15% FBS in a 5% CO 2–95% air humidified atmosphere
at 37 ◦C.
Cell proliferation assay
Analysis of cell proliferation was performed with cell counting kit-8 (Dojindo, Japan) assay. CXCR4 inhibitor WZ811 and mTOR inhibitor everolimus were pur- chased from Selleck (Huston, TX) and dissolved in DMSO. The treatment of WZ811 was performed as 5 lM, 10 lM, 20 lM, 40 lM and 80 lM, while the
working concentration of everolimuse was 5 nM, 10 nM, 20 nM, 40 nM and 80 nM. Cells were seeded in 96-well plate at a concentration of 1 × 105/mL. After 48 h, 10 uL cell counting kit-8 were added to each well and incubated for 2 h. The absorbance at 450 nm was measured by a microplate reader. Growth inhib- ition was calculated by the formula (O.D absorbance of treatment group – O.D absorbance of blank)/(O.D absorbance of control group – O.D absorbance of blank) × 100%. The synergetic effect of two drugs was measured by combination index (CI) using CalcuSyn
software (Biosoft, Cambridge, UK). A CI value of <1 indicated synergy.
Western blot
Antibody against CXCR4 was purchased from Abcam (Cambridge, UK). Antibodies against phospho-mTOR S2448(p-mTOR), phospho-p70S6K T389 (p-p70S6K) and phospho-4E-BP1 T37/46 (p-4E-BP1) were purchased from Cell Signaling Technology (Beverly, MA).
All the cell lines were treated with WZ811 and ever- olimus in single way or combination for 72 h. Then, the samples in each group were collected and sepa- rated by standard SDS-PAGE gel electrophoresis and transferred to NC membrane. Blocked with 5% nonfat milk supplemented in 0.1% TBST, membrane was probed with primary antibodies at 4 ◦C overnight.
After washed with TBST, membrane was incubated with HRP-conjugated secondary antibodies (Santa Cruz, CA) for one hour. The bands were detected and quantified in Image Lab software (Bio-Rad Laboratories, CA).
Immunohistochemistry
Paraffin-embedded sections were cut 3 lm thick, fixed in formalin and incubated in anti-mTOR (phospho S2448, 1:50 dilution, Abcam) and anti-CXCR4 (UMB2- IHC-antibody, 1:75 dilution, Abcam, Cambridge, UK), which are mouse antihuman monoclonal antibodies. They were then incubated with multiuse secondary antibody (1:1000 dilution, Dako, UK). Staining was per- formed using an EnVisionTM Peroxidase/DAB Rabbit/ Mouse Detection Kit (Dako, UK). At high magnification,10 representative fields of view, CXCR4 and mTOR showed identifiable positive tumor cells with moderate (golden brown) or strong (brown) intensity. Other cells stained very weakly (grayish), and these cells were con- sidered negative. Reactive hyperplasia served as a posi- tive control and PBS as a negative control. According to the method established by Hans [17], CD10 (1:80, Novocastra, Newcastle, UK), BCL-6 (1:10, Dako, Glostrup,ImageCXCR4 AND MTOR EXPRESSION IN PATIENTS WITH DLBCL 3Downloaded by [Australian Catholic University] at 06:43 29 September 2017Denmark) and MUM1/IRF4 (1:40, Dako, Glostrup, Denmark) were used to determine the DLBCL subtype. All stained sections were judged by two pathologists on the same day. When their conclusions differed, a final decision was reached by consensus.
Response to treatment
All patients underwent a median of six cycles R-CHOP therapy without any dose adjustments at 21-day inter- val. Efficacy was evaluated once per month and after six courses of treatment. All patients underwent a CT scan of the abdomen, pelvis, chest and neck, regard- less of the site of the onset of the disease. Criteria from the International Working Group (International Workshop criteria) were used to divide the response to treatment into complete remission (CR), clinical complete remission (CRU), partial remission (PR), stable disease (SD) and disease progression (PD) [18]. The total effective rate was defined as CR, Cru, and PR. The primary endpoints of the present work were progres- sion-free survival (PFS) and overall survival (OS). Progression-free survival was defined as the time elapsed from the effective start of treatment to pro- gression, relapse, death or the last follow-up. Overall survival was defined as the time elapsed from the ini- tial diagnosis to death or the last follow-up.
Statistical analysis
Correlations between protein expression and clinicopa- thological variables were assessed using Fisher’s exact test. Correlations between CXCR4 and mTOR were assessed using the nonparametric Spearman r correl- ation coefficient. The Kaplan–Meier method was used to estimate survival functions. They were compared using the log-rank test. Survival analyses were assessed using Cox’s proportional hazard regression model. The hazard ratio and 95% confidence interval
were also estimated. Here, p values were based on two-sided tests and p < .05 was considered significant. The statistical analyses were performed using SPSS
version 18 (SPSS Inc., Chicago, IL).
Results
Expression of CXCR4 and mTOR in DLBCL
A total of 56 patients with pathological tissue speci- mens were examined, including 32 men and 24 women, aged 26–87 years (mean age, 60 years), the clinicopathological features of 56 patients with DLBCL was summarized in Table 1. The immunohistochemical
Clinicopathological features of 56 patients with DLBCL.
Clinicopathological features n % Age
¼
60 or younger (n 32) 32 57
¼
Older than 60 (n 24) 24 43
Sex
¼
Male (n 32) 32 57
¼
Female (n 24) 24 43
ECOG PS
¼
Lower than 2 (n 27) 27 48
¼
2 or higher (n 29) 29 52
Stage
¼
I or II (n 27) 27 48
¼
III or IV (n 29) 29 52
Extranodal sites
¼
1 site (n 45) 45 80
¼
More than 1 site (n 11) 11 20
LDH
Normal (n ¼ 36) 36 64
IPIHigh (n ¼ 20) 20 36
¼
0–2 (n 33) 33 59
¼
3–5 (n 23) 23 41
Subtype
GCB (n ¼ 27) 27 48
Non-GCB (n ¼ 29) 29 52
ECOG: Eastern Cooperative Oncology Group; IPI: International Prognostic Index; LDH: lactate dehydrogenase PS: performance status.
Hans algorithm was used to determine the GCB/non-GCB cases.results of CXCR4 and mTOR were shown in Figure 1. There were 34 CXCR4-positive (60.7%) and 31 mTOR- positive samples (55.4%). CXCR4-positive cells were stained light brown, distributed in the membrane and cytoplasm. mTOR-positive cells were stained brown, distributed in the cytoplasm and the perinuclear area. Correlation analysis was used to determine whether CXCR4 or mTOR markers had any association witheach other in DLBCL. Results indicated that CXCR4 was positively correlated with mTOR expression (r ¼ 0.602; p < .01; Table 2).
CXCR4 and mTOR expression in DLBCL patients
The relationship between CXCR protein expression and clinical features is shown in Table 3. The presence of CXCR4 was found to be closely associated with presen- tation in patients that showed high levels of lactatedehydrogenase (LDH) level (p ¼ .009), high IPI score (p ¼ .030) and non-GCB subtype (p ¼ .006). No associ- ation was detected between the expression of CXCR4 and age, sex, stage, number of extranodal sites, per- formance status or recurrence (p > .05). As indicated in Table 4, there was no association between the expres-sion of mTOR and age, sex, stage, number of extrano- dal sites, performance status, immunophenotypic subtype, LDH level or recurrence (p > .05).
Thereslightly but not significantly more mTOR-positivepatients in the high-risk group than in the low-risk group (p ¼ .589).Z.-Z. XU ET AL. Immunohistochemistry study of CXCR4 and mTOR expression on paraffin tissue sections of patients with DLBCL (original magnification 40) (a) H&E stain presenting diffuse large neoplastic cells. (b) Negative control. (c) CXCR4 with diffuse cytoplasmic and membrane staining of neoplastic cells. (d) mTOR with both cytoplasmic and perinuclear accentuation of neoplastic cells.
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Table 2. Correlation between CXCR4 and mTOR expression.
Variable CXCR4 positive CXCR4 negative Total p value Total 34 22 56 .000
mTOR positive 27 4 31
mTOR negative 7 18 25
The overall response (OR) rate was 78.6% (44/56) for the entire population. The expression of CXCR4 was found to be negatively correlated with remission. The overall response rate was 64.7% (22/34) for the CXCR4-positive cohort of patients, and 100% (22/22)for those who were CXCR4 negative (p ¼ .002; Table 3).
Similarly, the expression of mTOR was negatively cor- related with the remission rate (p ¼ 0.047; Table 4).
Correlation between CXCR4, mTOR protein expression and patient survival
At the time of analysis, 18 (32%) patients were dead, and 38 (68%) patients remained alive as of last con- tact. For the surviving patients, the overall median fol- low-up period was 24 months (range, 11–61 months). The association between the expression of CXCR4 and mTOR protein and the survival analysis is shown in Figure 2. Survival analysis showed that patients with positive expression of CXCR4 had shorter survival time (estimated 3-year PFS, 29.3% vs. 49.0%; P ¼ 0.008; esti- mated 3-year OS, 35.1% vs. 49.1%; p ¼ .010) (Figure. 2(A–B)). The expression of mTOR was also significant in predicting both inferior PFS (estimated 3-year, 32.2% vs. 42.5%; p ¼ .039) and OS (estimated 3-year, 32.3% vs. 51.1%; p ¼ .036) (Figure 2(C–D)). Multivariate analysis covered age, performance status, LDH IPI, stage, subtype, CXCR4 and mTOR, and results indi- cated no significant association between CXCR4 or mTOR expression and survival. The IPI remained a sig- nificant prognostic factor for PFS (p ¼ .024) and OS(p ¼ .030)
Synergistic effect of CXCR4 inhibitor WZ811 combined with the mTOR inhibitor everolimus in DLBCL cell lines
The DLBCL cell lines SUDHL-4 and Ly-3 were cultured with various concentrations of WZ811 or everolimus for 48 h. WZ811 inhibited the proliferation of the cell lines in a dose-dependent manner with a 50% inhibi- tory concentration (IC 50) of 61 lM for SUDHL-4 and 27 lM for Ly-3. A similar effect was observed inImageCXCR4 AND MTOR EXPRESSION IN PATIENTS WITH DLBCL 5
Clinical characteristics and CXCR4 expression status of patients with DLBCL.
CXCR4 þ CXCR4 —
Clinicopathological features n % n % p
Age .788
¼
60 or younger (n 32) 20 36 12 21
¼
Older than 60 (n 24) 14 25 10 18
Sex 1.000
¼
Male (n 32) 19 34 13 23
¼
Female (n 24) 15 27 9 16
ECOG PS .274
¼
Lower than 2 (n 27) 14 25 13 23
¼
2 or higher (n 29) 20 36 9 16
Stage .585
¼
I or II (n 27) 15 27 12 21
¼
III or IV (n 29) 19 34 10 18
Extranodal sites .226
1 site (n ¼ 45) 28 50 21 38
Clinical characteristics and mTOR expression status of patients with DLBCL.
mTOR þ mTOR —
Clinicopathological features n % n % p
Age .59
¼
60 or younger (n 32) 19 34 13 23
¼
Older than 60 (n 24) 12 21 12 22
Sex .42
¼
Male (n 32) 16 29 16 29
¼
Female (n 24) 15 26 9 16
ECOG PS 1.00
¼
Lower than 2 (n 27) 15 27 12 21
¼
2 or higher (n 29) 16 29 13 23
Stage 1.00
¼
I or II (n 27) 15 27 12 21
¼
III or IV (n 29) 16 29 13 23
Extranodal sites .226
¼
1 site (n ¼ 45) 26 46 23 41
¼
More than 1 site (n 11) 6 11 1 1
LDH .009ω
¼
Normal (n 36) 17 30 19 34
¼
High (n 20) 17 30 3 6
IPI .030ω
¼
0–2 (n 33) 16 29 17 30
¼
3–5 (n 23) 18 32 5 9
Subtype .006ω
¼
GCB (n 27) 11 20 16 29
¼
Non-GCB (n 29) 23 41 6 10
Recurrence .458
¼
No (n 47) 27 48 20 36
¼
Yes (n 9) 7 13 2 3
Treatment response .002ω
CR (n ¼ 44) 22 39 22 39
PD(n ¼ 12) 12 22 0 0
More than 1 site (n 11) 5 9 2 4
LDH .78
¼
Normal (n 36) 19 34 17 30
¼
High (n 20) 12 22 8 14
IPI .589
¼
0–2 (n 33) 17 30 16 29
¼
3–5 (n 23) 14 25 9 16
Subtype .178
¼
GCB (n 27) 12 21 15 27
¼
Non-GCB (n 29) 19 34 10 18
Recurrence .716
¼
No (n 47) 25 44 22 40
¼
Yes (n 9) 6 11 3 5
Treatment response .047ω
CR (n ¼ 44) 21 38 23 41
PD(n ¼ 12) 10 18 2 3
CR: complete response; ECOG: Eastern Cooperative Oncology Group; IPI: International Prognostic Index; LDH: lactate dehydrogenase; PD: progres- sive disease; PS: performance status.
Hans algorithm was used to determine the GCB/non-GCB cases.
p values were calculated using Fisher’s exact test.
ωBold values represent p < .05.
CR: complete response; ECOG: Eastern Cooperative Oncology Group; IPI: International Prognostic Index; LDH: lactate dehydrogenase; PD: progres- sive disease; PS: performance status.
Hans algorithm was used to determine the GCB/non-GCB cases.
p values were calculated using Fisher’s exact test.
ωBold values represent p < .05.
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everolimus-treated cell lines with an IC 50 of 46nM for SUDHL-4 and 25 nM for Ly-3. The effects of com- bination treatment with WZ811 and everolimus were also determined (Figure 3(A)). Cell lines were treated with different doses of WZ811 (5, 10, 20, 40, 80 lM) and everolimus (5, 10, 20, 40, 80 nM) for 48 h.
Analysis using CalcuSyn software showed that the CIs for most combinations were <1 (Figure 3(B)), indicat- ing synergistic effects on inhibiting the growth of
DLBCL cells.
To further determine the mechanism of synergistic effect of drug combination, the expression of CXCR4 and the mTOR-signaling pathway were assessed by western blot. Coexposure with WZ811 (20 lM) and everolimus (20 nM) for 72h caused significant down- regulation of CXCR4 and p-mTOR in comparison with each single agent alone, and consequently phosphor- ylation of p70S6K and 4E-BP1 were also decreased. Thus, the rituximab/everolimus combination displayed enhanced effects on suppressing CXCR4/mTOR signal- ing and the expression of its regulated proteins (Figure 4).
Discussion Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous lymphoid malignancy. It is the most common subtype of non-Hodgkin’s lymphoma, typic- ally found in adults. It also has one of the highest mortality rates in the developed world [19]. DLBCL is usually aggressive and characterized by rapidly grow- ing tumors in the spleen, lymph nodes, bone marrow, liver and other organs [1]. DLBCL usually involves malignant progression and poor prognosis.
C-X-C chemokine receptor type 4 (CXCR4) is a che- mokine receptor specific to CXCL1 2 (SDF-1). The CXCL12/CXCR4 axis plays an important role in many physiological processes, including trafficking and homeostasis of immune cells and homing and reten- tion of hematopoietic stem cells within the bone mar- row [20]. CXCR4 can activate various intracellular signaling transduction pathways and downstream effectors that mediate cell survival, proliferation, chemotaxis, migration and adhesion [21]. CXCR4 may be an indicator of metastasis and poor prognostic
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6 Image Z.-Z. XU ET AL.
Image Figure 2. Progression-free survival (a) and overall survival (b) according to CXCR4 expression in patients with DLBCL. Progression- free survival (c) and overall survival (d) according to mTOR expression in patients with DLBCL.
Table 5. Multivariate analysis for factors affecting PFS and OS.
PFS OS
Factor p value HR 95% CI p value HR 95% CI
Older age .332 1.728 0.572–5.223 .618 1.336 0.428–4.164
ECOG (2-4) .650 0.663 0.112–3.911 .921 0.916 0.164–5.126
Stage (III/IV) .809 0.831 0.186–3.713 .936 0.941 0.215–4.129
LDH high .500 1.583 0.416–6.021 .263 2.201 0.554–8.750
IPI (3–5) .024 19.023 1.473–245.665 .030 15.999 1.304–196.289
Subtype (non-GCB) .708 1.260 0.376–4.222 .473 1.578 0.454–5.482
CXCR4 (positive) .672 1.508 0.225–10.122 .126 3.152 0.725–13.699
mTOR (positive) .368 2.001 0.442–9.047 .870 0.863 0.147–5.051
CI: confidence interval; ECOG: Eastern Cooperation Oncology Group; HR: hazard ratio; IPI: International Prognostic Index; PFS: progression-free survival. Log–rank test was used for univariate analysis.
Bold values represent p < .05.
CXCR4 AND MTOR EXPRESSION IN PATIENTS WITH DLBCL Image 7
Image
Figure 3. Combination of WZ811 and everolimus treatment synergistically inhibited cell growth in DLBCL cell lines. (a) Indicated cell lines were seeded in 96-well plates and then treated for 48 h with different concentrations of WZ811, everolimus and their respective combinations as indicated. Cell growth inhibition was evaluated using CCK8 assay. (b) Combination indexes (CIs) were calculated using CalcuSyn software. Data are means of three independent experiments; bars, SD.
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Image
Figure 4. Combination of rituximab and everolimus downre- gulated CXCR4-/mTOR-signaling pathway in DLBCL cell lines. Cells were treated with WZ811 (20 lM) alone, everolimus (20 nM) alone or WZ811 plus everolimus for 72 h. Levels of CXCR4, p-mTOR, p-p70S6K and p-4E-BP1 were analyzed by Western blotting. b-actin was used as a loading control.factor in several hematologic and nonhematologic malignancies. Overexpression of CXCR4 has been found to be associated with poor prognosis in acute myelogenous leukemia (AML) [22]. Similar observations have been made in other hematological cancers such as myelodysplastic syndrome (MDS) [23] and acute lymphoblastic leukemia (ALL) [24], in which CXCR4 expression on cancer cells was found to contribute to therapeutic resistance [25–26]. In 20 patients with non-Hodgkin lymphomas, a significant decrease in CXCR4 mRNA expression was observed in the BM after treatment. This decrease was found to be correlated with a significantly lower risk of death [27].
Recently, some studies have successfully shown that CXCR4 receptor has a pivotal role in the dissemination of DLBCL [11–13]. CXCR4 expression was found to be cor- related with disease progression in 12 cases of primary testicular DLBCL [11], and poor survival in 94 DLBCL cases [12]. It has also been found to independently predict the progression of germinal center B-cell-like diffuse large B-cell lymphoma [13]. However, in a study of Asian patients, CXCR4 was not associated with sur- vival for DLBCL patients [28].
In this study, the expression CXCR4 and their clin-
ical significance were investigated in 56 Chinese patients with DLBCL. Results showed that CXCR4 was expressed in 60.7% of the DLBCL samples collected. CXCR4 was found to be closely associated with more frequent presentation in patients whose lactate
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8 Image Z.-Z. XU ET AL.dehydrogenase (LDH) levels (p ¼ .009) and IPI scores were high (p ¼ .030). Results have shown that CXCR4 was significantly associated with non-GCB subtype (p ¼ .006). These results are consistent with those of a previous study in which CXCR4 was one of the genesthat defined the signature to cluster the ABC and GCB DLBCL subgroups [29]. Data collected here indicate that the expression of CXCR4 was closely correlated with a lower response rate (p ¼ .002) and a shorter PFS(p ¼ .008) and OS (p ¼ .010), which was consistent withfindings reported by Moreno MJ and Chen J [12–13]. The prognostic role of CXCR4 has shown clinical prom- ise for immunochemotherapy, and these findings must be confirmed using larger cohorts and, ideally, pro- spective, randomized clinical trials.
Treatment with AMD3100, a CXCR4 antagonist, can increase the rate of antibody-mediated cell death in disseminated lymphoma models. This suggests that CXCR4 agonists may play a role in B-cell-targeted ther- apy for the treatment of B-cell malignancies in clinical settings [30]. CXCR4 expression is positively regulated by the developmental signaling pathways such as oncogenic pathways phosphoinositide 3-kinase (PI3K)/AKT [31], NF-jB and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) [32]. The activation of the SDF-1/CXCR4 signaling activates MAPKs signaling, which promotes chemotaxis and pro-liferation, induces phospholipase C (PLC)/protein kin- ase C (PKC)-Ca2 þ signaling promoting cell migration and affects PI3K-/AKT-promoting cell survival [33].
This indicates a positive feedback loop between CXCR4 and the signaling pathways mediating tumorigenicity of cancer cells. The target of rapamycin (mTOR) kinase in mammals is an essential mediator of growth signaling. It originates from PI3K, and the activation of mTOR by Akt can foster cell proliferation and survival by regulat- ing critical molecules such as cyclin D1 [34]. The con- stitutive activation of the PI3K/AKT/mTORC1 pathway in GCB-DLBCL plays a central role in promoting sur- and resistance to chemotherapy and represents a rational therapeutic target in relapsed or refractory GCB-DLBCL [35]. Inhibition of target of rapamycin com- plex 1 (mTORC1) and PI3K blocks proliferation and induces cell death in BCR-subtype of ABC-DLBCL [36]. The relationship between the immunohistochemical expressions of mTOR and the clinical outcome was investigated in patients with DLBCL. Results indicate that mTOR was abnormally expressed in 55.4% of DLBCL and coexpressed with CXCR4 in 48% of the samples. Spearman r correlation analysis showed CXCR4 to be positively correlated with mTOR expres- sion. The expression of mTOR was negatively corre- lated with the remission rate. Although the expression of CXCR4 and mTOR was not associated with survival for DLBCL patients as indicated by multivariate analysis due to the limited size of the examined DLBCL cohort, our results suggested that the CXCR4 and mTOR sig- naling cascades may play a pivotal role in the progres- sion and dissemination of DLBCL.
We further analyzed the antiproliferative efficacy of the CXCR4 inhibitor WZ811 alone or combined with the mTOR inhibitor everolimus in DLBCL cell lines. We observed that the growth suppression effect of WZ811 and everolimus was significantly increased when both drugs were used in combination, relative to the effects of monotherapy. This combination strategy exerted an enhanced effect on inhibition of CXCR4/mTOR signal- ing and the expression of its regulated proteins, thus provide preclinical evidence and a theoretical basis for this novel strategy for treating DLBCL.
In conclusion, the results reported here indicate that CXCR4 was expressed in 60.7% of DLBCL and positively correlated with mTOR expression. Strong CXCR4 expression was associated with high levels of lactate dehydrogenase (LDH) and high IPI scores with non-GCB subtypes. The expression of CXCR4 and mTOR were found to be negatively correlated with remission. Kaplan–Meier analysis indicated a significant decrease in the rate of progression-free survival (PFS) and in that of overall survival (OS) in patients positive for CXCR4 and mTOR expression. The combination therapy with CXCR4 inhibitor WZ811 and mTOR inhibi- tor everolimus showed synergistic effect in DLBCL cell lines. The current results indicate that CXCR4 and mTOR expression may be suitable for use as bio- markers in the prediction of prognosis in patients with DLBCL and development of novel therapeutic strategies.
Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article online at https://doi.org/10.1080/10428194.2017.1379077.
Funding
This work was partly supported by National Natural Sciences Foundation of China (81600151), Shanghai Municipal Bureau of Health and Family Planning Commission (Grants 20154Y0168, Shanghai, China) and the Program for Outstanding Young Health Personnel of Huangpu District of Shanghai (Grants RCPY1409, Shanghai, China).
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