The prognostic role of combining Krüppel-like factor 4 score and grade of inflammation in a Norwegian cohort of oral tongue squamous cell carcinomas

Abstract

Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor involved in inflammation, cancer development, and progression. However, the relationship between KLF4, inflammation, and prognosis in oral cancer is not fully understood. KLF4 expression levels were examined in a multicenter cohort of 128 oral squamous cell carcinoma (OSCC) specimens from the tongue (OTSCC) using immunohistochemistry. In two external KLF4 mRNA datasets (The Cancer Genome Atlas/The Genotype-Tissue Expression Portal), lower KLF4 mRNA expression was found in OSCC and head and neck squamous cell carcinomas (HNSCC) than in control oral epithelium. These data indicate that down-regulation of KLF4 mRNA is linked to OSCC/HNSCC progression. Using Cox-multivariate analysis, a significantly favorable 5-year disease-specific survival rate was observed for a subgroup of patients with a combination of high levels of KLF4 expression and inflammation. OSCC cell lines exposed to IFN-γ showed a significant upregulation of nuclear KLF4 expression, indicating a link between inflammation and KLF4 expression in OSCC. Overall, the current data suggest a functional link between KLF4 and inflammation. The combination of high KLF4 nuclear expression and marked/moderate stromal inflammation might be useful as a favorable prognostic marker for a subgroup of OTSCC patients.

Keywords: KLF4; cancer of the tongue; stromal cells; survival.

FIGURE 1

KLF4 mRNA levels in head and neck squamous cell carcinoma (HNSCC) and oral squamous cell carcinoma (OSCC). (A) KLF4 mRNA levels in HNSCC and paratumor control epithelium. GEPIA box plot analysis tool [44]. (B) KLF4 mRNA expression in OSCC and normal control epithelium (Student's t‐test)

FIGURE 2

Immunohistochemical staining of oral tongue squamous cell carcinoma (OTSCC). A high (A) and low (B) nuclear KLF4 expression in representative OTSCC. Immunopositivity is seen in brown. Hematoxylin counterstain. Scalebar = 50μm

FIGURE 3

Kaplan–Meier plots (log rank test) of 5‐year disease‐specific survival in oral tongue squamous cell carcinomas with regard to the presence of KLF4 and inflammation. (A) A trend was seen for high KLF4 expression and favorable 5‐year disease‐specific survival (p = 0.064). (B) Patients with marked/moderate inflammation in the tumor stroma had a significantly better disease‐specific survival as compared with patients with slight or no inflammation (p = 0.026), (all tumors n = 128). (C,D) The combination of different KLF4 and inflammation score identified 105 patients of which survival data were available for 91. Here, a subgroup of patients with a high nuclear KLF4 expression (KLF4^high) and a marked/moderate stromal inflammation (inflammation^high) demonstrated a significantly favorable disease‐specific survival as compared to patients with other combinations of KLF4/inflammation (p = 0.006)

FIGURE 4

The influence of IFN‐γ on KLF4 nuclear expression. Oral cancer cells (CaLH3 and PE/CA‐PJ49 clone E10) were immunostained with anti‐KLF4 antibody (green) and incubated with DAPI (blue) for nuclear localization (six technical replicates). (A,B) Before IFN‐γ exposure. (C,D) Following 24 h exposure to IFN‐γ. Scale bar = 50μm. (E,F) In both cell lines, a statistically significant increase in KLF4 expression was found after IFN‐γ exposure, as shown by KLF4 positive area/total nuclear area (E), and mean pixel intensity (F)