Glypican-1, -3, -5 (GPC1, GPC3, GPC5) and Hedgehog Pathway Expression in Oral Squamous Cell Carcinoma

Abstract

Proteoglycans are involved in tumor development and may regulate the Hedgehog (HH) pathway. This study aimed to investigate the gene and protein expression of glypican-1 (GPC1), -3 (GPC3), and -5 (GPC5) in oral squamous cell carcinoma (OSCC) and tumor-free lateral margins (TM) and their association with the HH pathway. Quantitative PCR was performed for GPC1, GPC3, GPC5, SHH, PTCH1, SMO, and GLI1 genes in samples of OSCC (n = 31), TM (n = 12), and non-neoplastic oral mucosa (NNM) of healthy patients (n = 6), alongside an immunohistochemical evaluation of GPC1, GPC3, and GPC5 proteins and HH proteins SHH and gliomaassociated oncogene homolog 1 (GLI1). Double staining for GPC3/ SHH, GPC5/SHH, GPC3/tubulin [ac Lys40], GPC5/Tubulin [ac Lys40], and GPC5/GLI1 was also performed. Overexpression of GPC1 and GPC5 in tumor samples and underexpressed levels of GPC3 gene transcripts were observed when compared with TM (standard sample). HH pathway mRNA aberrant expression in OSCC samples and a negative correlation between GPC1 and GPC5 at transcription levels were detected. GPC1 staining was rare in OSCC, but positive cells were found in NNM and TM. Otherwise positive immunostaining for GPC3 and GPC5 was observed in OSCCs, but not in NNM and TM. Blood vessels adjacent to tumor islands were positive for GPC1 and GPC5. Co-localization of GPC3-positive and GPC5-positive cells with SHH and Tubulin [ac Lys40] proteins was noted, as well as of GPC5 and GLI1. The absence of the GPC1 protein in neoplastic cells, underexpression of the GPC3 gene, and co-localization of GPCs and HH proteins may indicate the maintenance of aberrant HH pathway activation in OSCC.

Identification and validation of microtubule depolymerizing agent, CYT997, as a potential drug candidate for hepatocellular carcinoma

“…Schlaepfer et al. reported the co‐localization of glypican‐3 (GPC3) and acetylated tubulin in oral squamous carcinoma cells 25 . We observed correlated down‐regulation of both GPC3 and acetylated α‐tubulin in three GPC3 expressing HCC cell lines treated with CYT997.…”

“…Schlaepfer et al reported the co-localization of glypican-3 (GPC3) and acetylated tubulin in oral squamous carcinoma cells. 25 We observed correlated down-regulation of both GPC3 and acetylated α-tubulin in three GPC3 expressing HCC cell lines treated with CYT997. Liu et al reported that Dactolisib (PI3K and mTOR inhibitor) and paclitaxel (a tubulin polymerizing agent) alone reduced the expression of GPC3, and combination treatment significantly reduced the expression of GPC3 in HCC in vivo.…”

“…The down‐regulation of β‐tubulins by CYT997 was substantially rescued when HepG2 cells were pretreated with proteasome inhibitor MG132, indicating that CYT997 down‐regulated β‐tubulins through the proteasomal degradation system (Figure 6B). Because of the association of glypican‐3 (GPC3) with acetylated tubulin, 25 we also studied the effect of CYT997 on GPC3 expression in HCC cells. Immunoblotting showed that treatment with CYT997 down‐regulated the expression of GPC3 (except in SNU449 cells which are GPC3 negative), as well as that of β‐catenin, and c‐Myc (Figure 6A).…”