STOML2 as a novel prognostic biomarker modulates cell proliferation, motility and chemo-sensitivity via IL6-Stat3 pathway in head and neck squamous cell carcinoma

Abstract

STOML2 (Stomatin-like protein 2) is up-regulated and acts as an oncogenic protein in multiple cancers. However, the role and regulatory mechanism of STOML2 in head and neck squamous cell carcinoma remain unclear. Here, we found that STOML2 is overexpressed and indicates poor outcomes in HNSCC. In addition, the expression of STOML2 correlates positively with T stage, lymph node metastasis and recurrence. Reduced STOML2 dramatically inhibits cell proliferation, colony formation and motility of HNSCC cells in vitro. Furthermore, the sensitivity of HNSCC cells towards cisplatin is obviously improved in STOML2-silencing cells. Subsequent studies suggest that STOML2 could regulate the expression of IL6 transcriptionally and then further induce the phosphorylation of Tyr705 residue of Stat3, whose activation plays a critical role in HNSCC. Taken together, these results for the first time demonstrate that STOML2 promotes HNSCC progression through activating IL6-Stat3 pathway and provide a promise for diagnosis and treatment for HNSCC.

Keywords: HNSCC; STOML2; Stat3; chemo-sensitivity; invasion; proliferation.

Figure 1

STOML2 expression level is increased in HNSCC and indicates poor prognosis. (A) The frequency of STOML2 somatic alteration across various cancers including HNSCC. (B) The analysis of GEO database (GSE25099 and GSE37991) indicated that the mRNA level of STOML2 was enhanced in HNSCC tissues. N, adjacent normal tissues, C, HNSCC tissues. (C) Representative photographs of IHC results of STOML2 at the invasive margin and the invasive front of HNSCC tissues. (D) Representative photographs of IHC results of STOML2 in HNSCC with different stages of differentiation. Scale bar in (C and D), 100 μm. (E) Kaplan-Meier survival curve showed that HNSCC patients (n=91) with high STOML2 expression level had an unfavorable overall survival (P=0.0379).

Figure 2

STOML2 expression in HNSCC cell lines. A. The mRNA level of STOML2 was measured in a panel of HNSCC cells by real-time PCR. B. The protein expression of STOML2 was detected in a panel of HNSCC cells by immunoblots. C. Three distinct siRNAs were introduced into both SCC25 and SCC15 cells, respectively. The STOML2 expressions in these cells were measured via real-time PCR and western blotting. Data, mean ± SD, **P<0.01.

Figure 3

STOML2 knockdown suppresses proliferation of HNSCC cells. A. The expression of STOML2 in SCC25 and SCC15 cells transfected with a pool of siRNAs against STOML2. B. Growth curve suggested that reduced STOML2 significantly inhibited cell proliferation. Data, mean ± SD, *P<0.05. C. Reduction of colony formation capacity in STOML2-silenced SCC25 and SCC15 cells. Data, mean ± SD, *P<0.05. D. STOML2 knockdown arrested cell cycle at S phase in SCC25 and SCC15 cells.

Figure 4

STOML2 knockdown inhibits motility of HNSCC cells in vitro. A. Representative photographs of gap at 0 hr and 24 hr of wound healing assay performed in HNSCC cells transfected with siRNAs against STOML2 or negative control. B. Transwell assay showed that depletion of STOML2 dramatically attenuated the abilities of migration and invasion of SCC25 and SCC15 cells. Scale bar, 100 μm. Data, mean ± SD, *P<0.05, **P<0.01. C. GEO database revealed that MMP9 correlated positively with STOML2. D. Western blotting results showed that STOML2 depletion reduced expression of MMP9.

Figure 5

Reduced STOML2 enhances sensitivity of HNSCC cells to cisplatin in vitro. (A) STOML2 deletion decreased cisplatin IC50 in SCC25 (from 17.08 to 4.161 μM) and SCC15 cells (from 15.12 to 3.847 μM). (B and C) Silenced STOML2 sensitized HNSCC cells to cisplatin treatment and increased apoptosis using flow cytometry (B) and colony formation assay (C). CDDP, cisplatin. Data, mean ± SD, *P<0.05, **P<0.01. (D) Depletion of STOML2 enhanced the level of cleaved Caspase 3 in the case of cisplatin dosing.

Figure 6

STOML2 regulates IL6-Stat3 pathway in HNSCC cells. (A) WB results showed that knockdown of STOML2 obviously reduced p-Stat3 (Y705), but affected little on the level of p-Stat3 (S727). (B) Immunofluorescence staining indicated that STOML2 knockdown impeded the accumulation of nuclear p-Stat3. (C) The expression of STOML2 was assessed by western blotting after blocking Stat3 expression. (D) Real-time PCR analysis indicated that STOML2 depletion decreased IL6 mRNA expression. Data, mean ± SD, *P<0.05. (E and F) Western blotting assay revealed that IL6 induced the phosphorylation of Stat3 at Y705 residue (E) and STOML2 regulated the activation of Stat3 in an IL-6 dependent manner (F).

Figure 7

Stat3 blocking attenuates proliferation, motility and chemo-resistance of HNSCC cells. (A and B) Transfection with si-Stat3 significantly impaired the capacities of cell proliferation (A) and colony formation (B) of SCC25 and SCC15 cells. (C) Stat3 depletion inhibited migration and invasion of HNSCC cells in vitro compared with negative control. Scale bar, 100 μm. (D and E) Stat3 knockdown increased the sensitivity of HNSCC cells to cisplatin and promoted apoptosis using flow cytometry (D) and colony formation assay (E). CDDP, cisplatin.