Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

Abstract

Background: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC).

Patients and methods: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFbeta1, TGFbeta2, TGFbeta3 in oral SCC.

Results: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFbeta isoforms, we found that Smad2 mRNA and TGFbeta1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFbeta1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population.

Conclusion: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFbeta signaling should be better clarified in the future.

Figure 1

Smad mRNA expression in oral SCC: A. RPA representative assay. Lane 1: riboprobe; represented as pairs: T-tumor M- matched adjacent mucosa; L32: housekeeping gene. B. Boxplot representing Smad1-8 mRNA expression. Boxes: 25^th, 50^th and 75^th percentiles; bars: 10^th and 90^th percentiles; °: outlier values (1.5-3 box-lengths from 75^th percentiles); _*: extreme values (>3 box-lengths from 75^th percentiles).

Figure 2

Kaplan-Meier survival curves of oral cavity SCC patients grouped according to Smad expression. Patients were categorized as positive (above) or negative (equal or below) according to median Smad expression in tumors. Log Rank test was performed for curves comparison. In E and F, patients were grouped according to the co-expression of Smad2 and Smad6. Smad2⁺/Smad6^-: n = 13; Smad2⁺/Smad6⁺: n = 11; Smad2^-/Smad6^-: n = 13; Smad2^-/Smad6⁺: n = 11.

Figure 3

Kaplan-Meier survival curves of larynx SCC patients grouped according to Smad expression. Patients were categorized as positive (above) or negative (equal or bellow) according to median tumor expression. Log Rank test was performed for curves comparison.

Figure 4

Hypothesis scheme of Smad influence on survival. TGFβ1 upregulation is associated with tumor agressiveness and poor patient survival. Smad2 and Smad6 have opposite roles in this process. Full and dashed lines represent our own findings and previous published data, respectively.