Sam68 regulates cell proliferation and cell adhesion‐mediated drug resistance (CAM‐DR) via the AKT pathway in non‐Hodgkin's lymphoma

Abstract

Objectives

Sam68 (Src‐associated in mitosis 68 kDa), a substrate for tyrosine kinase c‐Src during mitosis, is up‐regulated in a variety of human cancers and acts oncogenically promoting tumour progression. This study has explored biological function and clinical significance of Sam68 in non‐Hodgkin's lymphoma (NHL).

Materials and methods

To examine Sam68 expression in NHL, clinically, eight diffuse large B‐cell lymphomas and four reactive lymphoid hyperplasia fresh‐frozen tissues were obtained for western blot and quantitative real‐time PCR analyses. Using immunohistochemical staining, paraffin wax embedded sections from 164 cases of NHL patients were used to evaluate prognostic value of Sam68. Cell Counting Kit‐8 (CCK‐8) and soft agar colony assays were conducted to investigate the role of Sam68 in cell viability and cell proliferation respectively. Furthermore, effects of Sam68 on cell adhesion‐mediated drug resistance (CAM‐DR) was determined by CCK‐8 assay and flow cytometric analysis.

Results

Expression status of Sam68 inversely correlated with clinical outcomes of patients with NHL, and it was also an independent prognostic factor for the outcomes. In addition, Sam68 was associated with proliferation of NHL cells. Knock‐down of its gene inhibited cell proliferation and colony formation by delaying cell cycle progression. Furthermore, OCI‐Ly8 and Jeko‐1 cells adhering to FN and HS‐5 expressed higher Sam68 protein, compared to their suspension counterparts. Sam68 promoted cell adhesion‐mediated drug resistance (CAM‐DR) via the AKT pathway.

Conclusions

Increased Sam68 expression in NHL resulted in poor prognosis, and it promoted CAM‐DR in NHL via AKT.

Abbreviations

Sam68

Src‐associated in mitosis of 68 kDa

NHL

non‐Hodgkin's lymphoma

RLH

reactive lymphoid hyperplasia

DLBCL

diffuse large B‐cell lymphoma

FL

follicular lymphoma

MCL

mantle cell lymphoma

MALT

extranodal lymphoma of mucosa‐associated lymphoid tissue

FN

fibronectin

CAM‐DR

cell adhesion‐mediated drug resistance

Introduction

Non‐Hodgkin's lymphomas (NHL) comprise a heterogeneous group of malignant lymphoproliferative disorders exhibiting a variety of types of clinical behaviour, biological phenotypes and prognoses 1. Incidence of NHL has been rising steadily over recent years, leading to significant numbers of deaths worldwide 2. Front‐line therapy for NHL has often involved radiation and chemotherapy, even though using these methods can induce high remission rates; relapse and drug resistance are common and contribute to low survival rates 3. Thus, it is important to explore relative mechanisms of drug resistance in NHL.

Many investigators have previously shown that the extracellular matrix (ECM) plays a pivotal role in cancer progression and response to therapy 4. Environment‐mediated drug resistance is made of two basic components: physical contact between tumour cells and microenvironmental elements [cell adhesion‐mediated drug resistance (CAM‐DR)] and soluble factor‐mediated drug resistance 5, 6. Mounting evidence now suggests that adhesion to fibronectin (FN)‐coated surfaces or bone marrow stromal cells can protect malignant lymphoma cells from apoptosis induced by chemotherapeutic drugs 7, 8. However, up to now the molecular mechanisms of these processes have been unclear.

Sam68 (Src‐associated in mitosis 68 kDa) belongs to the STAR (signal transduction and activation of RNA metabolism) family, which was initially identified as a Src‐associated substrate in mitosis 9, 10. Sam68 participates in a great variety of cellular processes including transcription, signal transduction, RNA metabolism, apoptosis and cell cycle progression 11. It is ubiquitously expressed, a series of published reports stating that Sam68 expression is up‐regulated in many human cancers, including prostate, cervical, renal cell and breast, indicating that Sam68 might act as an oncogene promoting tumour progression 12, 13, 14. However, deregulation of Sam68 in malignant human tissues has only been observed in a limited number of cancer types. As yet, it has been uncertain whether or not Sam68 has any clinical significance in NHL.

In this study, we demonstrated that expression status of Sam68 was inversely correlated to clinical outcomes of patients with NHL, and also that it was an independent prognostic factor for NHL. Furthermore, we showed that Sam68 regulated cell proliferation and CAM‐DR via the AKT pathway. Our findings shed new light on the important role of Sam68 in cancer development.

Materials and methods

Patients and specimens

Tumour specimens including 76 cases of indolent lymphoma and 88 cases of progressive lymphoma were obtained from the Department of Pathology, Affiliated Cancer Hospital of Nantong University (Nantong, China) from 1993 to 2009. These tissues were fixed in 10% buffered formalin and embedded in paraffin wax for histopathological diagnosis and immunohistochemical labellin. Histological NHL cases were classified according to World Health Organization (WHO) systems, and their detailed clinical information was obtained. Furthermore, eight pathologically confirmed DLBCL and four reactive lymphoid hyperplasia fresh‐frozen tissues from recent patients were obtained for western blot and quantitative real‐time PCR analyses. Written informed consent was obtained from each patient prior to tissue acquisition. Institutional approval was obtained from the Ethical Review Board of the Affiliated Cancer Hospital of Nantong University, prior to the study.

Immunohistochemistry staining and evaluation

Slides were cleared in xylene and rehydrated in ethanol–water solutions. Next, sections were heated at 120 °C for 3 min in 0.01 m citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase activity was blocked by immersion in 3% hydrogen peroxide for 20 min and tissues were blocked with universal blocking serum for 20 min (Dako Diagnostics, Carpinteria, CA, USA). Tissue sections were then incubated in anti‐Sam68 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Ki‐67 antibodies (1:100; Santa Cruz Biotechnology) for 2 h at room temperature, before being incubated for 20 min each in biotin‐labelled secondary antibody and streptavidin‐peroxidase (Dako Diagnostics). Finally, 3, 3′‐diaminobenzidine (DAB) was used for signal development. Tissue sections were dehydrated, mounted and evaluated by three observers, blinded to their clinical data, using a semiquantitative scoring system for both intensity of stain and percentage of positive malignant cells. These were scored as: 0 (0% tumour cells stained); 1 (0–25% tumour cells stained); 2 (25–50% tumour cells stained); and 3 (50–100% tumour cells stained). Intensity of staining was coded as follows: 0 (negative), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). Then, we multiplied the two scores and used the result as the staining index (SI), classifying them into two groups: low expression (0–3) and high expression (4–9).

RNA isolation and quantitative real‐time PCR (qRT‐PCR)

Total RNA was extracted from eight DLBCL and four reactive lymphoid hyperplasia fresh‐frozen tissues using Trizol reagent (Gibco BRL, Gaithersburg, MD, USA). Approximately 1 μg RNA was used for reverse transcription with oligo‐dT (18T) (Invitrogen, Carlsbad, CA, USA). cDNA was amplified using the following primers: forward 5′‐ATG CAG CGC CGG GAC GAC‐3′ and reverse 5′‐TTA ATA ACG TCC ATA TGG GTG‐3′. Quantitative real‐time PCR was carried out in triplicate with SYBR Green PCR Master Mix using a 7900HT qPCR system thermal cycler (Applied Biosystems, Foster City, CA, USA). GAPDH mRNA was used as internal control for each sample, and CT value for each sample was normalized to GAPDH mRNA.

Western blot analysis and antibodies

Western blotting was performed according to methods described previously 15. All antibodies used were purchased from Santa Cruz Biotechnology except the following: AKT (Cell Signaling Technology, Beverly, MA, USA), Gsk‐3β (Cell Signaling Technology, Beverly, MA, USA), p‐AKT (Cell Signaling Technology) and p‐Gsk‐3β (Cell Signaling Technology).

Cell culture and transient transfection

Human MCL, Jeko‐1, DLBCL, OCI‐Ly8 cell lines were obtained from Fudan University (Shanghai, China) and cells were grown in RPMI1640 (Sigma‐Aldrich, St. Louis, MO, USA), supplemented with 10% foetal bovine serum. Human bone marrow stromal cell line HS‐5, was obtained from Cell Library, China Academy of Science, and cultured in F12 (Sigma‐Aldrich) with 10% foetal bovine serum at 37 °C and 5% CO₂. Sam68 siRNA was designed and synthesized by GenechemCo (GenechemCo. Ltd, Shanghai, China). Four siRNA sequences targeting Sam68 were used: si‐1, 5′‐ACC GGA TAT GAT GGA TGA T ‐3′; si‐2, 5′‐ACA AGG GAA TAC AAT CAA A‐3′; si‐3, 5′‐TGA GGA GAA TTA CTT GGA T‐3′; and si‐4, 5′‐CGG CAG AAA TTG AGA AGA T‐3′. Full‐length Sam68 cDNA was amplified from a human lymphoma library using PCR, and subcloned into a pcDNA3.1 construct (Invitrogen). Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Cell co‐culture

First, dishes were coated with HS‐5 cells or 40 μg/ml human FN (Sigma‐Aldrich) at 37 °C. Secondly, lymphoma cells (10⁵cells/ml) were allowed to adhere to pre‐incubated HS‐5 or FN for 24 h. Lastly, adherent cells were carefully removed for subsequent experiments.

Cell Counting Kit‐8 (CCK‐8) assay

Cells were seeded into 96‐well plates at 10⁵ cells/well. CCK‐8 reagents (Dojindo, Kumamoto, Japan) were added to each well at indicated times, and incubated at 37 °C for 1 h. Absorbance at 450 nm was read using an automated plate reader.

Soft agar colony assays

Cells were resuspended at 1 × 10³ in 500 μl of 0.35% agar solution containing cell culture medium, and layered on top of 0.8% agar layers, in six‐well plates, which were then maintained for 14 days at 37 °C with 5% CO₂. Cell colonies were stained with 0.5% crystal violet and visualized by microscopy.

Flow cytometric analysis

To analyse cell cycles, cells were fixed in 70% methanol at −20 °C overnight, then incubated with 1 mg/ml RNase A for 30 min and stained with 50 mg/mL of propidium iodide in PBS. Data acquisition and analysis were performed in a Becton‐Dickinson FACS Calibur flow cytometer (Becton‐Dickinson, San Jose, CA, USA). To analyse apoptosis, flow cytometric assay was performed using an Annexin V: FITC Apoptosis Detection Kit I (Becton‐Dickinson) according to the manufacturer's protocol.

Statistical analysis

Calculations were analysed using the Statistical Package for Social Sciences SPSS 19.0 software (SPSS Inc., An IBM Company, Chicago, IL, USA). All data shown represent results of at least three independent experiments; P value <0.05 was deemed statistically significant.

Result

Sam68 expression in RLH and DLBCL tissues

To determine the role of Sam68 in NHL, we first examined mRNA expression of Sam68 in four RLH and eight DLBCL samples, using qRT‐PCR. This revealed that Sam68 mRNA expression was clearly higher in DLBCLs than in RLH tissues (Fig. 1a). Meanwhile, western blot assays were performed to investigate Sam68 protein expression. Consistent with its mRNA level, Sam68 protein expression was also higher in DLBCL compared to RLH (Fig. 1b).

Figure 1.

Expression of Sam68 in RLH and DLBCL tissues. (a) Sam68 mRNA expression in four RLH and eight DLBCL tissues was detected by real‐time PCR. CT value of GAPDH was subtracted from CT value of Sam68. (b) Analysis of Sam68 protein expression in four RLH and eight DLBCL tissues by western blotting. Histogram demonstrates the ratio of Sam68 protein to β‐actin. The experiment was repeated at least 3 times. *P < 0.05.

Correlation between high expression of Sam68 and clinicopathological variables

To further confirm the relationship between Sam68 expression and clinicopathological variables, 164 cases of NHL, including 76 indolent lymphomas and 88 progressive lymphomas, were used to investigate Sam68 expression using immunohistochemical staining. The result showed that Sam68 was significantly lower in indolent lymphomas, such as follicular lymphoma (FL) and extranodal lymphoma of mucosa‐associated lymphoid tissues (MALT), compared to those of progressive lymphoma, such as DLBCL and Burkitt lymphoma (P < 0.001; Table 1, Fig. 2). Furthermore, Sam68 expression in NHL tissues significantly correlated with clinicopathological features, such as Ki‐67 expression and lymphoma category (P < 0.001; Table 2). However, there was no correlation with age or gender. Kaplan–Meier analysis revealed that patients with high expression of Sam68 had a poorer overall survival (Fig. 3), and multivariate Cox regression analysis indicated that Sam68 expression was an independent prognostic factor (HR = 1.767, 95% CI 1.095–2.853; Table 2).

Table 1.

Relationship between expression levels of Sam68 and clinicopathological features of 164 NHL specimens

Variables	Low expression		High expression		χ2	P Value
	n	%	n	%
Age (years)
≤60	36	54.55	30	45.45	2.557	0.110
>60	41	41.84	57	58.16
Gender
Male	38	43.18	50	56.82	1.083	0.298
Female	39	51.32	37	48.68
B symptom
Negative	16	50.00	16	50.00	0.148	0.700
Positive	61	46.21	71	53.79
LDH
Low	37	44.58	46	55.42	0.380	0.538
High	40	49.38	41	50.62
Ann Arbor stage
I–II	48	48.98	50	51.02	0.402	0.526
III–IV	29	43.94	37	56.06
Ki‐67 LI
Low	70	82.35	15	17.65	88.790	<0.001
High	7	8.86	72	91.14
Lymphoma category
Progressive	26	29.55	62	70.45	23.098	<0.001
Indolent	51	67.11	25	32.89

Statistical analyses were performed by the Pearson χ² test.

P value <0.05 was considered significant.

LDH, lactate dehydrogenase; Ki‐67 LI, Ki‐67 labelling index.

Figure 2.

Representative images of immunohistochemical staining. Expression of Sam68 was significantly lower in indolent lymphoma, compared to progressive lymphomas. Omission of primary antibody served as negative control.

Table 2.

Multivariate Cox regression analysis of Sam68 and clinical variables predicting survival from 164 NHL specimens

Variablesa	HR (95% CI)	P Value
Age	1.255 (0.861–1.831)	0.237
Gender	0.899 (0.634–1.276)	0.552
B symptom	0.974 (0.614–1.545)	0.911
LDH	0.826 (0.583–1.171)	0.284
Ann Arbor stage	0.869 (0.592–1.274)	0.471
Ki‐67 LI	2.493 (1.500–4.145)	<0.000
Lymphoma category	0.587 (0.392–0.879)	0.010
Sam68	1.767 (1.095–2.853)	0.020

^aCoding of variables: age was coded as 0, ≤60 years and 1, >60 years; gender was coded as 0, male and 1 female; B symptom was coded as 0, negative and 1, positive; LDH was coded as 0, low and 1, high; Ann Arbor stage was coded as 0, I–II and 1, III–IV; Ki‐67 LI was coded as 0, low and 1, high; lymphoma category was coded as 0, progressive lymphoma and 1, indolent lymphoma; Sam68 expression was coded as 0, low and 1, high.

P value <0.05 was considered significant.

Figure 3.

Kaplan–Meier survival curve in 164 NHL patients according to Sam68 expression. Log rank test, P < 0.05.

Sam68 promoted proliferation of NHL cells via regulating cell cycle progression

As Sam68 correlated with Ki‐67 expression (P < 0.001; Table 1; Fig. 2), a proliferation marker, we speculated that Sam68 correlated with cell proliferation. To validate our hypothesis, Jeko‐1 and OCI‐Ly8 cells were transiently transfected with Sam68‐siRNA or control‐siRNA. After transfection, efficiencies of Sam68‐siRNA‐mediated down‐regulation were confirmed by western blot analyses (Fig. 4a). This showed that Sam68‐siRNA#4 exhibited superior efficiency of the four siRNA sequences used. Thus, Sam68‐siRNA#4 was chosen for subsequent assays. Next, CCK‐8 assays were performed to measure cell viability of Sam68 knock‐down cells. This showed that Sam68 knock‐down resulted in significant inhibition of cell viability (Fig. 4b). Colony formation assays showed that down‐regulation of Sam68 clearly reduced proliferation of Jeko‐1 and OCI‐Ly8 cells (Fig. 4c). In addition, flow cytometric analysis showed that Sam68‐siRNA#4 increased percentages of cells in G0/G1 phase but reduced percentages of cells in S phase, suggesting that Sam68 promoted cell proliferation by regulating cell cycle progression (Fig. 4d).

Figure 4.

Sam68 regulating proliferation of NHL cells. (a) Jeko‐1 and OCI‐ly8 cells were transfected with either control‐siRNA or Sam68‐siRNA. Efficiency of Sam68‐siRNA‐mediated down‐regulation was confirmed by western blotting using β‐actin as loading control. (b) Jeko‐1 and OCI‐ly8 cells were transfected with either control‐siRNA or Sam68‐siRNA#4 at the indicated time, then CCK‐8 assays were performed to evaluate cell viability. (c) Soft agar colony assays were performed to evaluate the effect of Sam68 on cell proliferation. Colony numbers were normalized to number of colonies formed by cells expressing control‐siRNA. (d) Jeko‐1 and OCI‐ly8 cells were transfected with either control‐siRNA or Sam68‐siRNA, then flow cytometric analysis was performed to analyse cell cycle progression. *;^# P < 0.05.

Sam68 promoted cell proliferation and inhibited apoptosis by enhancing phospho‐AKT expression

It has previously been reported that Sam68 regulates proliferation of breast cancer via the AKT pathway 14. Thus, we asked whether Sam68 would also promote NHL cell proliferation by regulating AKT. To test that hypothesis, OCI‐Ly8 and Jeko‐1 cells were transfected with Sam68‐siRNA#4 or control‐siRNA. Western blot assays showed that Sam68 knock‐down resulted in a reduced expression of p‐AKT and p‐GSK‐3β, but not in total AKT and GSK‐3β (Fig. 5a). Furthermore, overexpression of Sam68 in OCI‐Ly8 and Jeko‐1 cells increased p‐AKT and p‐GSK‐3β expression (Fig. 5b). AKT inhibitor (MK2206) was then used to check the role of AKT in the proliferative effect of Sam68. As shown in Fig. 5c, MK2206 significantly reduced p‐AKT and p‐GSK‐3β expression in OCI‐Ly8 and Jeko‐1 cells. Intriguingly, Sam68‐mediated promotion of cell proliferation was eliminated by MK2206 in both OCI‐Ly8 and Jeko‐1 cells (Fig. 5d). Flow cytometric assays were used to investigate the effect of Sam68 on apoptosis. This showed that there were higher proportions of apoptotic cells in Sam68‐siRNA#4‐transfected cells compared to control‐siRNA‐transfected ones. Moreover, Sam68‐siRNA#4‐mediated apoptosis was augmented when treated with MK2206. As expected, overexpression of Sam68 reduced apoptosis; however, anti‐apoptotic effects of Sam68 were abrogated in MK2206‐treated cells (Fig. 5e). All data collectively suggest that Sam68 promoted cell proliferation and inhibited apoptosis by enhancing phospho‐AKT expression.

Figure 5.

Sam68 promotion of cell proliferation and inhibition of apoptosis by enhancing phospho‐ AKT expression. (a) Jeko‐1 and OCI‐ly8 cells were transfected with either control‐siRNA or Sam68‐siRNA#4, then cells were lysed and analysed for AKT, Gsk‐3β, p‐AKT and p‐Gsk‐3β expression by western blotting. (b) Jeko‐1 and OCI‐ly8 cells were transfected with either myc‐control or myc‐Sam68, then cells were lysed and analysed for AKT, Gsk‐3β, p‐AKT and p‐Gsk‐3β expression by western blotting. (c) OCI‐Ly8 and Jeko‐1 cells were treated with AKT inhibitor MK2206 or DMSO (control), then analysed for AKT, Gsk‐3β, p‐AKT and p‐Gsk‐3β expression by western blotting. (d) Jeko‐1 and OCI‐ly8 cells were transfected with either myc‐control or myc‐Sam68, then treated with MK2206 or DMSO; then soft agar colony assays were performed to evaluate cell proliferation. (e) Jeko‐1 and OCI‐ly8 cells were transfected with either Sam68‐siRNA#4 or myc‐Sam68, or their respective control, then treated with MK2206 or DMSO, and flow cytometric analysis was performed to analyse apoptosis. *; ^# P < 0.05.

Sam68 promoted cell adhesion‐mediated drug resistance in NHL

To investigate the role of Sam68 in CAM‐DR, we constructed a specific cell adhesion model. Western blot assays showed that adhesion of OCI‐Ly8 and Jeko‐1 cells to FN and HS‐5 cells increased Sam68 protein expression, compared to their suspension counterparts (Fig. 6a). CCK‐8 assays showed that adhesion to FN and HS‐5 cells significantly protected OCI‐Ly8 and Jeko‐1 cells from cytotoxicity compared to those cultured in suspension. Interestingly, these effects were attenuated when transfected with Sam68‐siRNA#4 (Fig. 6b). Furthermore, flow cytometric analysis was used to further determine Doxo‐induced apoptosis in FN‐adherent cells. Similarly, it showed that there were more Doxo‐induced apoptotic cells in Sam68‐siRNA#4‐transfected cells compared to controls (Fig. 6c). All data suggested that Sam68 played an important role in the CAM‐DR phenotype. To determine whether Sam68 regulated CAM‐DR phenotype via the AKT pathway, OCI‐Ly8 and Jeko‐1 cells were treated with MK2206 or DMSO. CCK8 assays revealed that Sam68‐mediated drug resistance was clearly reversed in MK2206‐treated cells (Fig. 6d,e). All these findings indicated that Sam68 promoted CAM‐DR via the AKT pathway.

Figure 6.

Sam68 promotion of CAM ‐ DR via the AKT pathway. (a) OCI‐Ly8 and Jeko‐1 cells were allowed to adhere to FN or HS‐5 cells or cultured in suspension. Cells were lysed and analysed for Sam68 expression by western blotting. (b) OCI‐Ly8 and Jeko‐1 cells were transfected with either control‐siRNA or Sam68‐siRNA#4, then cells were cultured in three different conditions including adhesion to FN, HS‐5 cells or cultured in suspension along with addition of 1 μM Doxo. CCK‐8 assay was performed to evaluate cell viability. (c) Flow cytometric analysis was used to determine Doxo‐induced apoptosis in FN‐adherent cells. (d,e) Jeko‐1 and OCI‐ly8 cells were transfected with either myc‐control or myc‐Sam68, then treated with MK2206 or DMSO, then cells were allowed to adhere to FN or HS‐5 cells or cultured in suspension along with addition of 1 μM Doxo. CCK8 assays were then performed to evaluate effects of myc‐Sam68 and MK2206 on CAM‐DR in OCI‐Ly8 and Jeko‐1 cells. *; ^# P < 0.05.

Discussion

A series of published reports has previously confirmed that the role of Sam68 is complicated. It participates in regulating cancer‐specific splicing events such as CD44, BCL‐X, cyclin D1 and SF2/ASF, involved in cell proliferation, cell cycle progression, apoptosis, cell migration and invasion 16, 17, 18, 19, 20. Some studies have indicated that Sam68 can act as a tumour suppressor. Sam68 down‐regulation is associated with ability to form metastatic tumours in nude mice and its deficiency leads to neoplastic transformation of murine fibrosarcomas in nude mice 21. Yet in contrast, many recent reports have demonstrated that Sam68 acts as an oncogene in many cancers. In cervical neoplasia, expression of Sam68 is significantly up‐regulated, and it promotes cell motility and invasion 12. In prostate cancer, Sam68 is up‐regulated, and reduced Sam68 delays cell cycle progression and reduces proliferation of prostate cancer cells 13. Up‐regulation of Sam68 also correlates with shorter patient survival in endometrial carcinoma and non‐small cell lung cancer 22, 23.

However, it is largely unknown whether Sam68 is involved in NHL development. In this study, we demonstrated that expression status of Sam68 inversely correlated with clinical outcomes of patients with NHL, and it was also an independent prognostic factor for NHL. Sam68 was associated with proliferation of NHL cells and knock‐down of its gene inhibited cell proliferation and colony formation by delaying cell cycle progression.

It has been widely demonstrated that tumour microenvironments influence tumour growth, metastasis and response to chemotherapy in NHL, multiple myeloma and hepatocellular carcinoma 6, 24, 25. Resistance to traditional chemotherapy is an obstacle to successful NHL therapy, thus it is necessary to study molecular mechanisms of CAM‐DR. Zhang et al. 26 reported that Sam68 interacted directly with the p85 regulatory subunit of PI3K and mediated PI3K/AKT activation during EV71 infection. Silencing Sam68 dramatically abrogated AKT phosphorylation. In peripheral blood mononuclear cells, leptin stimulation promotes tyrosine phosphorylation of Sam68, which leads to activation of PI3K via associating with p85 27. Sanchez‐Margalet et al. 28 reported that Sam68 was associated with p120GAP, upon tyrosine phosphorylation by the insulin receptor and recruited GAP to the PI3K pathway. Sam68 may localize to the cytoplasm where it interacts with PI3K and induces lymph node metastasis through the AKT/GSK‐3β/Snail pathway‐mediated epithelial‐mesenchymal transition in cervical cancer 12. In breast cancer cells, lack of Sam68 suppresses cell proliferation and anchorage‐independent growth by augmentation of p21^Cip1 and p27^Kip1, as well as elevated FOXO transcriptional activity and deactivation of the PI3K/AKT pathway, leading to G1–S phase arrest 14. It is well documented that down‐regulation of Sam68 sensitizes prostate cancer cells to apoptosis induced by DNA‐damaging agents. This led us to test whether Sam68 would modulate cell proliferation and CAM‐DR phenotype in NHL via AKT. Our study revealed that Sam68 promoted proliferation and inhibited apoptosis of NHL cells by enhancing phospho‐AKT expression. Interestingly, Sam68 appeared to have specific effects on CAM‐DR via the AKT pathway. However, whether other mechanisms exist underlying Sam68‐mediated drug sensitivity in NHL needs to be further investigated.

In sum, our study has shown that Sam68 was highly associated with cell proliferation and played an important role in CAM‐DR in NHL. In the future, this may be an effective strategy for treatment of NHL.

Conflict of interest

There are no potential conflicts of interest to disclose.