ASC contributes to metastasis of oral cavity squamous cell carcinoma

Abstract

ASC (Apoptosis-associated Speck-like protein containing a CARD) acts as a platform protein in the inflammasome cascade of some cancer types. However, its potential involvement in OSCC (oral cavity squamous cell carcinoma) has not yet been determined. Here, we investigated the potential role of ASC in OSCC. RT-qPCR analysis of 20 paired tumor and adjacent normal tissue samples revealed that the mRNA levels of ASC, along with IL-1β, CASP1, and NLRP3 in ASC-associated NLRP3 inflammasome were significantly elevated in OSCC tissues. Immunohistochemical staining of these four proteins in 111 clinical specimens revealed that high-level expression of ASC was significantly associated with tumor stage, node stage (p=0.001), overall stage (p<0.001), extracapsular spread (p<0.001), perineural invasion (p=0.004) and tumor depth (p<0.001). Kaplan-Meier survival analysis further revealed that high-level ASC expression was correlated with poorer overall survival (p=0.001), disease-specific survival (p<0.001) and disease-free survival (p<0.001). Studies using OSCC cell lines indicated that high-level ASC expression enhanced cell migration and invasion, and experiments using an orthotropic nude mouse model confirmed that ASC overexpression induced metastasis of OSCC cells. This is the first report to show that ASC contributes to OSCC metastasis, and that high-level ASC expression is a marker for poor prognosis in OSCC patients.

Keywords: ASC; OSCC; metastasis.

Figure 1. Inflammasome proteins are highly expressed in OSCC

A. Microarray results for NLRP3 inflammasome-associated proteins. B. Relative mRNA expression levels of asc, il-1b, casp1, and nlrp3 in tumor (T) and adjacent normal tissues (N). C. ASC, IL-1β, CASP1, and NLRP3 proteins are overexpressed in the tumor regions (T) compared to adjacent normal regions (N) of OSCC clinical samples. Magnification, X400; bar, 50 μm. D. Kaplan-Meier survival analysis of OSCC patients. The overexpressions of ASC, IL-1β, and CASP1 (but NLRP3) are associated with poor prognoses for OS (upper), DSS (middle) and DFS (lower). ASC positive (+), n=54; ASC negative (-), n=57; IL-1β positive (+), n=35; IL-1β negative (-), n=76; CASP1 positive (+), n=50; CASP1 negative (-), n=61.

Figure 2. ASC promotes migration and invasion ability in OSCC cell lines

A and B. Cell proliferation assays were performed on SAS cells overexpressing ASC or the control vector (A) or transiently transfected with control or ASC-targeting siRNAs (B) Western blot results show the expression levels of ASC in the SAS cells. C. Migration and invasion assays of ASC-overexpressing SAS cells show 2.3- and 6-fold increases in cell migration (top) and invasion (bottom), respectively. D. Migration and invasion assays of ASC-knockdown cells show ~75% decreases in migration (top) and invasion (bottom) compared with the vector control.

Figure 3. ASC promotes tumor metastasis in an animal model

A. Design of the animal study. B. The IVIS visualization system used to track tumor growth and metastasis. Mice were inoculated with SAS_Luc2 or SAS_Luc2_ASC on day 1, and images were monitored and recorded on days 8, 15 and 22. Red arrows indicate the metastatic lymph node. C. Comparison of metastatic lymph node between SAS_Luc2 and SAS_Luc2_ASC mice, data shown as mean±SE from 3 independent experiments; 5 mice were tested in each group for each experiment (p=0.033). D. Image of lymph node metastasis in the cervical region of a SAS_Luc2_ ASC mouse. Red arrows indicate the metastatic lymph node. E. Histological image of tongue and lymph nodes sections in four representing SAS_Luc2_ASC-carrying mice, and in two SAS_Luc2 control mice as described in Results. ASC is highly expressed in SAS_Luc2_ASC mice, but not in SAS_Luc2 mice. Magnification, X200; bar, 100μm.