AFF4 promotes tumorigenesis and tumor-initiation capacity of head and neck squamous cell carcinoma cells by regulating SOX2

Abstract

Super elongation complex (SEC) controls gene transcription by releasing Pol II from pausing. Previous studies have shown that dysfunction of SEC was associated with multiple human cancers, such as leukemia and breast cancer. However, the role of SEC in head and neck squamous cell carcinoma (HNSCC) development remains largely unknown. In this study, we found expression of AF4/FMR2 family member 4 (AFF4), the core component of SEC, was upregulated dramatically in HNSCC cell lines and tumor tissues. By using siRNA-mediated depletion and overexpression of AFF4, we demonstrated AFF4 promoted proliferation, migration and invasion of HNSCC cells. Moreover, we found AFF4 enhanced the aldehyde dehydrogenase (ALDH) activity and sphere formatting activity and was required for the tumor-initiation capacity of stem-like cells in HNSCC cell lines. Mechanistically, we found the role of AFF4 in regulation of HNSCC cell behaviors was mainly mediated by sex-determining region Y box2 (SOX2), a critical regulator involved in development of several human cancers. SOX2 expression changed in parallel with AFF4 expression in response to depletion and overexpression of AFF4, respectively. More importantly, overexpression of SOX2 rescued the inhibited proliferation, migration, invasion and ALDH activity induced by knockdown of AFF4 in HNSCC cells, at least in part. Collectively, our findings indicate AFF4 may serve as a biomarker and a potential target of therapies for patients with HNSCC.

Figure 1.

AFF4 is upregulated in HNSCC. (a, b) qPCR analysis of SEC component expression in SCC1 cells (a), SCC23 cells (b). The fold-change was normalized to the matched expression in HaCaT cells. n = 3. (c, d) Western blot analysis of AFF4 expression in HaCaT cells, SCC1 cells (c) and SCC23 cells (d). The a-tubulin was used as a loading control. n = 3. (e) Representative images of immunohistochemistry staining with AFF4 in normal human tongue mucosa and HNSCC tissues. Bar indicates 50 μm. (f) AFF4 expression was upregulated in HNSCC tissues (n = 69), compared with normal human tongue mucosa (n = 10). (g, h) AFF4 expression was correlated with tumor progression from TNM stage 1,2 (T1,2, n = 35) to TNM stage 3,4 (T3,4, n = 26) and from Grade 1 (G1, n = 16) to Grade 2, 3 (G2,3, n = 49). *P-value < 0.05; **P-value < 0.01.

Figure 2.

AFF4 promotes proliferation of HNSCC cells. (a, b) SCC1 and SCC23 cells were transfected with scrambled control siRNA (SCR) and siAFF4, respectively. qPCR analysis (a) and western blot (b) was conducted to examine of AFF4 expression at 48 h post transfection. n = 3. (c, d) MTT assay results showed knockdown of AFF4 inhibited cell proliferation in SCC1 cells (c) and SCC23 cells (d). n = 3 at each time point. (e, f) SCC1 and SCC23 cells were transfected with retroviruses expressing HA-tagged AFF4. Cells transfected with empty vector were used as a control. n = 3. (e) Western blot and qPCR analysis (f) were conducted to examine expression of HA and AFF4 at 48 h post transfection respectively. n = 3. (g, h) MTT assay results showed overexpression of AFF4 enhanced cell proliferation in SCC1 cells (g) and SCC23 cells (h). n = 3 at each time point. *P-value < 0.05; **P-value < 0.01.

Figure 3.

AFF4 enhances tumor-initiation capacity of HNSCC cells. (a, b) Flow cytometry assay performed to examine the effects of AFF4 depletion on ALDH activity of SCC1 cells (a) and SCC23 cells (b). Cells stained with ALDH+DEAB, an ALDH inhibitor, served as the negative control. (c) Quantification of ALDH⁺ cells in a and b. n = 3. (d) Sphere formation assay conducted to examine the effects of AFF4 depletion on sphere formation capacity of SCC1 cells and SCC23 cells. Bar indicates 200 μm. (e) Quantification of formed spheres (diameter exceeding 10 µm) in d. n = 3. (f) qPCR analysis of the effects of AFF4 depletion on expression of BMI1, CD44, NANOG and SOX2 in SCC1 cells. n = 4. (g) In vivo limiting dilution assay conducted to examine the effects of AFF4 depletion on tumor-initiation capacity of SCC1 cells in vivo. n = 6 for each group. Total mice, 48. (h) Quantification of formed tumors in g. (i, j) Flow cytometry assay performed to examine the effects of AFF4 overexpression on ALDH activity of SCC1 cells (i) and SCC23 cells (j). (k) Quantification of ALDH⁺ cells in i and j. n = 3. (l) Sphere formation assay conducted to examine the effects of AFF4 overexpression on sphere formation capacity of SCC1 cells and SCC23 cells. Bar indicates 200 μm. (m) Quantification of formed spheres (diameter exceeding 10 µm) in k. n = 3. (n) qPCR analysis of the effects of AFF4 overexpression on expression of BMI1, CD44, NANOG and SOX2 in SCC1 cells. n = 4. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.

Figure 4.

AFF4 promotes migration and invasion of HNSCC cells. (a, b) Wound-healing assay performed to assess the effects of AFF4 depletion on migration activity of SCC1 cells (a) and SCC23 cells (b). Bar indicates 400 μm. (c) Quantification of migration distance in a and b. n = 3. (d) Transwell assay conducted to study the effect of AFF4 depletion on invasion activity of SCC1 cells and SCC23 cells. Bar indicates 200 μm. n = 3. (e, f) Wound-healing assay performed to assess the effects of AFF4 overexpression on migration activity of SCC1 cells (e) and SCC23 cells (f). Bar indicates 400 μm. (g) Quantification of migration distance in e and f. n = 3. (h) Transwell assay conducted to study the effect of AFF4 overexpression on invasion activity of SCC1 cells and SCC23 cells. Bar indicates 200 μm. n = 3. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.

Figure 5.

AFF4 promotes SOX2 expression and recruits CDK9 to SOX2 promoter. (a) Western blot of SOX2 expression in response to depletion of AFF4 at 48 h post transfection. n = 3. (b) Western blot of SOX2 expression in response to overexpression of AFF4 at 48 h post transfection. n = 3. (c, d) ChIP assay was conducted to study the recruitment of AFF4 at SOX2 promoter in SCC1 cells (c) and SCC23 cells (d) transfected with SCR and siAFF4 at 48 h post transfection. n = 3. (e, f) ChIP assay was conducted to study the recruitment of CDK9 at SOX2 promoter in SCC1 cells (e) and SCC23 cells (f) transfected with SCR and siAFF4 at 48 h post transfection. n = 3. (g, h) ChIP assay was conducted to study the H3K4me3 levels at SOX2 promoter in SCC1 cells (g) and SCC23 cells (h) transfected with SCR and siAFF4 at 48 h post transfection, respectively. n = 3. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001.

Figure 6.

Overexpression of SOX2 rescued the inhibitory effects on HNSCC cell behavior induced by AFF4 depletion. (a) Western blot analysis of SOX2 expression in SOX2-overexpressing SCC1 and SCC23 cells in response to AFF4 knockdown at 48 h post transfection. n = 3. (b, c) MTT assay for cell proliferation in SOX2-overexpressing SCC1 cells (b) and SCC23 cells (c) in response to AFF4 knockdown at 3, 5 days. n = 3 at each time point. (d) Flow cytometry assay for ALDH activity of SOX2-overexpressing SCC1 cells in response to AFF4 knockdown at 48 h post transfection. n = 3. (e) Sphere formation assay conducted to evaluate sphere formation capacity of SOX2-overexpressing SCC1 and SCC23 cells in response to AFF4 knockdown after 2 weeks of culture in suspension. Bar indicates 200 μm. n = 3. (f) Would healing assay for migration activity of SOX2-overexpressing SCC1 cells in response to AFF4 knockdown. n = 3. (g, h) Transwell assay for invasion activity SOX2-overexpressing SCC1 and SCC23 cells in response to AFF4 knockdown. n = 3. *P-value < 0.05; **P-value < 0.01.