HIF‐1α mediates CXCR4 transcription to activate the AKT/mTOR signaling pathway and augment the viability and migration of activated B cell‐like diffuse large B‐cell lymphoma cells

Diffuse large B‐cell lymphoma (DLBCL) is the most common lymphoid malignancy with a high relapse rate. We previously found that C‐X‐C motif chemokine receptor 4 (CXCR4) was highly expressed in DLBCL and associated with poor prognosis. This study focused on the effect of hypoxia‐inducible factor‐1α (HIF‐1α) on CXCR4 expression and the DLBCL progression. Two activated B cell‐like DLBCL cell lines Ly‐3 and SUDHL2 were transfected with overexpression and knockdown plasmids or HIF‐1α. The viability and migration of DLBCL cells were significantly increased under hypoxic conditions, or upon HIF‐1α overexpression under normoxic conditions, but the HIF‐1α downregulation led to inverse trends. However, the promoting effects of HIF‐1α overexpression on DLBCL cells were suppressed by Plerixafor (a CXCR4 inhibitor). The luciferase and chromatin immunoprecipitation assays revealed that HIF‐1α bound to the functional site HRE1 on CXCR4 promoter to activate its transcription. HIF‐1α‐mediated CXCR4 activation further led to increased phosphorylation of AKT/mTOR under hypoxic conditions. Taken together, this work reports that HIF‐1α promotes viability and migration of activated B cell‐like cells under hypoxia, which might involve the transcription of CXCR4 and the activation of the AKT/mTOR pathway. The finding may provide novel lights in the management of DCBCL.


| BACKGROUND
Non-Hodgkin's lymphoma is responsible for 260,000 deaths in 2020, comprising 2.8% of worldwide cancer death according to GLOBO-CAN 2020, 1 in which diffuse large B-cell lymphoma (DLBCL) accounting for 30%-40%. 2 According to the cell-of-origin (COO) classification, DLBCL can be identified as activated B-cell-like (ABC), germinal center B-cell-like (GCB), and unclassified subtypes. The heterogeneity of DLBCL is reflected in the genetic differences among all subtypes. 3 The addition of rituximab to the standard chemotherapy regimen of cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) significantly improved the survival time of DLBCL patients. However, a considerable part of these patients (30%-40%) suffered from refractory or eventually relapsed disease after R-CHOP, which had become the main reason for the poor prognosis of DLBCL. 4 Therefore, it is of great significance to find the key molecules that regulate the tumor progression and control the growth and migration of tumors to enhance the efficacy of DLBCL treatment and to improve the prognosis of patients.
The overgrowth of malignant tumors leads to the development of a hypoxic microenvironment, which impacts the biological process and prognosis of tumors. Hypoxia-inducible factor-1 (HIF-1), a nuclear transcription factor regulating hypoxia response, strongly correlates with tumor metastasis, angiogenesis, poor prognosis of patients, drug resistance, and prognosis of patients. 5 HIF-1 is a heterodimer synthesized by polymerization of HIF-1α and HIF-1β subunits whose expression is strictly controlled by the level of intracellular oxygen concentration. However, HIF-1α is extremely easy to degrade under normoxia. Under hypoxia, HIF-1α transports into the nucleus and combines with the hypoxic response element (HRE) upstream of the target gene promoter, which plays an essential regulatory role in tumor proliferation, apoptosis, energy metabolism, migration, and chemotherapy resistance. 6 Although the progression of cancer is closely related to hypoxia, the regulatory mechanism of HIF-1α is not identical, which needs to further explore in DLBCL.
As a family of small molecule cytokines proteins, chemokines, and their receptors have differential expression and unbalanced regulatory mechanisms in a variety of tumor cells, suggesting that they play an essential role in tumorigenesis, development, migration, and metastasis, among which chemokine receptor type 4 (CXCR4) is in particular closely associated with tumors. 7 Our previous study showed that CXCR4 expression was significantly associated with high IPI score, non-GCB subtype and a significant decrease in progression-free survival (PFS) and overall survival (OS). 8 10 Therefore, we hypothesized that HIF-1α may affect the progression of DLBCL by acting on CXCR4 promoter region.
In addition, the AKT/mTOR pathway is one of the signal transduction pathways most closely related to cell proliferation and tumor progression. 11 Our previous study found that this signaling pathway is related to the occurrence and development of DLBCL, whose continuous activation is a poor molecular biological prognostic factor for DLBCL. 12 It has been found that knockdown of HIF-1α inhibited AKT/mTOR pathway in ovarian cancer. 13 Therefore, in present study, we investigated whether HIF-1α can promote DLBCL cells proliferation and migration by regulating CXCR4 and activating the AKT/mTOR pathway under hypoxia.

| Western blot assay
Western blot assay was used to analyze the expression of CXCR4 and AKT/mTOR pathway-related proteins. Total protein from cells was lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime) and the protein concentration was determined using a bicinchoninic acid kit (Beyotime). Next, the protein samples were After being washed three times with tris-buffered saline and Tween-20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 1 h at room temperature. The protein bands were visualized using an electrochemiluminescence system (Tanon) and analyzed using ImageJ.
Relative protein expression was quantified with GAPDH as the internal loading.

| Statistical analysis
All data in this study were analyzed with SPSS 20.0 software (SPSS) and presented as mean ± standard deviation. Comparisons between different experimental groups were performed by one-way analysis of variance (ANOVA) with Dunnett's test, or by two-way ANOVA with Turkey test. The values of p < 0.05 was considered significant.

| Efficiency of knockdown HIF-1α plasmids
The knockdown efficiency of three types of plasmids was detected by RT-qPCR and western blot assay to select the best knockdown plasmids for further experiments. As shown in Figure 1, the relative mRNA and protein levels of HIF-1α in cells in the three HIF-1α-ShRNA-pGIPZ plasmid groups were significantly lower than the control group (p < 0.05). The HIF-1α-Sh1 with the best knockdown efficiency (p < 0.01) was used as the knockdown plasmid for subsequent experiments.

| The overexpression of HIF-1α promoted the viability and migration of DLBCL cells
The viability and migration of DLBCL cells were detected by CCK8 and Transwell assays. As shown in Figure 2A 3.4 | HIF-1α bound to the functional site HRE1 of CXCR4 promoter to enhance the expression of CXCR4 Whether and how HIF-1α enhances the expression of CXCR4 was detected by dual-luciferase reporter assay and ChIP assay. As shown in Figure 3A, the expression of CXCR4 was significantly enhanced under normoxia with HIF-1α-OE and under hypoxia (p < 0.001).
Furthermore, two primer regions of the functional site HRE1 of CXCR4 promoter were used to detect whether HIF-1α acts on the CXCR4 promoter to affect its expression. Results from ChIP assays showed that hypoxia and HIF-1α-OE treatment significantly promoted the enrichment of CXCR4 promoters (p < 0.001, Figure 3B).

| HIF-1α regulated CXCR4 to affect the expression of AKT/mTOR pathway-related proteins
Activation of the Akt/mTOR has been reportedly linked to the oncogenic events mediated by CXCR4. 14 hypoxia (p < 0.001) ( Figure 4B). To further clarify the relationship between CXCR4 and AKT/mTOR pathway, we examined the activity of AKT/mTOR pathway in Ly3 and SUDHL2 cells. It was found that the proportion of phosphorylated AKT and mTOR was significantly decreased in cells after knockdown of CXCR4 ( Figure 4C).

| DISCUSSION
Diffuse large B-cell lymphoma accounts for 30%-40% of newly diagnosed non-Hodgkin lymphomas. Gene expression profiling has classified DLBCL into the GCB subtype, which is associated with a better prognosis, and ABC/non-GCB subtype, which has a more aggressive clinical evolution. 16  F I G U R E 4 (A) The expression of CXCR4 and AKT/mTOR pathway-related proteins were detected via western blot assay (**p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. the blank group under normoxia; ## p < 0.01, ### p < 0.001 vs. the blank group under hypoxia). (B) The expression of CXCR4, AKT/ mTOR pathway-related proteins were detected via western blot assay (**p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. the HIF-1α-OE group under normoxia; ### p < 0.001 and #### p < 0.0001 vs. the blank group under hypoxia). (C) The expression of CXCR4, AKT/mTOR pathway-related proteins were detected via western blot assay (**p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. the control group). AKT, protein kinase B; CXCR4, chemokine receptor type 4; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HIF-1α, hypoxia-inducible factor-1α; mTOR, mammalian target of rapamycin. SDF-1. At present, Plerixafor combined with granulocyte colony stimulating factor has been used in non-Hodgkin's lymphoma after autologous transplantation. 22 In the present study, Plerixafor was used to block the effect of CXCR4 to verify whether the effect of HIF-1α on DLBCL is related to the expression of CXCR4. Consistent with a previous study that the expression of CXCR4 in chronic lymphocytic leukemia cells is under the transcriptional control of HIF-1α, 23 our results demonstrated that Plerixafor inhibited the viability and migration of DLBCL cells cultured under hypoxia or transfected with HIF-1α-OE, which indicated that CXCR4 played a key role in the regulatory pathway of HIF-1α to DLBCL cells. Although this result indicates that HIF-1α regulates CXCR4, we cannot verify whether HIF-1α directly acts on CXCR4. Therefore, the mechanism of HIF-1α regulating CXCR4 was further studied through the dual luciferase reporter assay and ChIP assay. Results showed that HIF-1α activated the transcription of CXCR4 by directly binding to the functional HRE1 region of CXCR4 promoter, thereby regulating the expression of CXCR4, which is consistent with the previous study. 24 AKT/mTOR signaling pathway regulates many cellular processes, including cell survival, proliferation, and metabolism, which is not surprising to see AKT/mTOR pathway is dysregulated in many cancers and is associated with drug resistance. 25 It has been reported that AKT/ mTOR pathway inhibitors are effective for non-Hodgkin's lymphoma. 26 Based on a previous study that AKT/mTOR pathway is a potential important signaling pathway and an unfavorable prognostic factor of DLBCL, 12 we detected the relationship among HIF-1α, CXCR4, and AKT/ mTOR pathway in DLBCL cells via western blot assay. The results further confirmed that HIF-1α regulated the expression of CXCR4 and demonstrated that HIF-1α regulated CXCR4 to activate AKT/mTOR pathway. Interestingly, some studies have shown that AKT/mTOR pathway can regulate the metabolism of cancer cells by HIF-1α, 25 which means that HIF-1α regulates the expression of CXCR4 to activate AKT/ mTOR pathway under hypoxia, and it may lead to the increase of HIF-1α expression and further promote the migration and progress of DLBCL cells. Collectively, these findings demonstrate that CXCR4 is a potential prognostic biomarker in activated B cell-like DLBCL and exhibits oncogenic roles via PI3K/AKT/mTOR pathway activation. HIF-1α played an essential role in CXCR4-mediated DLBCL progression, indicating that HIF-1α and CXCR4 are candidate therapeutic targets for activated B celllike DLBCL ( Figure 5).

| CONCLUSION
This study demonstrated that HIF-1α bound with the functional site