SGSM2 inhibits thyroid cancer progression by activating RAP1 and enhancing competitive RAS inhibition

Abstract

Thyroid cancer (TC) is one of the most common malignancies involving the head and neck, and its incidences are increasing every year. Small G protein signaling modulators 2 (SGSM2) belongs to a newly identified protein group that contributes to numerous cancer progression. However, its role in TC remains unknown. The aim of this study was to explore the functions and underlying molecular mechanism of SGSM2 in the progression of thyroid tumorigenesis. Here, we demonstrated that SGSM2 expression was markedly decreased in TC, and that lower SGSM2 expression was potentially related to worse patient prognosis. Meanwhile, the SGSM2 levels were not directly correlated with BRAF or RAS mutations in TC. Based on our functional analysis, ectopic SGSM2 expression strongly prevented cell proliferation, migration, invasion, and tumorigenic activity in TC cells that harbored wild type RAS. Mechanistically, we demonstrated that SGSM2 interacted with Small G protein Ras-associated protein 1(RAP1) and augmented its activity. Activated RAP1 then competitively suppressed RAS activation and thereby downregulated output of MAPK/ERK and PI3K/Akt networks, which are primary contributors of TC. In summary, the present study reports a tumor suppressive role of SGSM2 in TC. Moreover, we revealed the underlying molecular mechanism, thus providing a potential therapeutic target for TCs that harbor wild type RAS.

Fig. 1. SGSM2 expression is decreased in primary thyroid cancer (TC).

a Relative SGSM2 mRNA levels was lower in PTC samples, relative to normal thyroid tissue (NTT) samples, 18 S RNA was used for normalization of qRT-PCR data. b SGSM2 was lower in PTC, compared to matched NTT in the TCGA cohort. c Relative SGSM2 mRNA levels in different subtypes of PTC samples from the TCGA dataset, ***, compared to Classical PTC, #, compared to Follicular PTC. d SGSM2 expression in PTC and matched NTT samples, as evidenced by Western blot. e SGSM2 protein expression (indicated by the red arrow) in epithelial cell was lower in PTC, compared to adjacent NNT samples. f SGSM2 expression was examined in various TC cell lines and immortalized thyroid epithelial cells via Western blot, GAPDH employed as loading control. g Relationship between SGSM2 expression and patient survival in PTC, median SGSM2 expression was employed as threshold. Data shown as mean ± SD. ***P < 0.001; */#P < 0.05.

Fig. 2. SGSM2 inhibits malignant biological properties of thyroid cancer (TC) cells.

a Ectopic SGSM2 levels in different TC cells, as evidenced by Western blot. SGSM2 overexpression significantly inhibited TC cell proliferation, as evidenced by EdU assay b and MTT assay c. d SGSM2 overexpression markedly suppressed colony formation of TC cells. SGSM2 overexpression markedly reduced migration c and invasion d of TC cells. Data shown as mean ± SD. ***P < 0.001; **P < 0.01; *P < 0.05.

Fig. 3. SGSM2 prevents tumor growth via suppression of both MAPK/ERK and PI3K/AKT networks in xenograft mouse models and in thyroid cancer (TC) cells.

a Stably expressing SGSM2 in the 8305 C cell line, as evidenced by Western blot. b The growth curve of xenografts derived from the 8305 C stably expressing SGSM2 or non-sense control (NC). c Images of dissected tumors. Box-whisker plot illustrates average tumor weight. Data expressed as mean ± SD (n = 5/group). d A typical Ki-67 staining of a dissected xenograft tumor shown in the left panel, and quantification of Ki-67 positive cells presented in the right. e Alterations in nude mice body weight. f The levels phosphor-ERK (p-ERK), and phosphor-AKT (p-AKT) were decreased in cells overexpressing SGSM2 and carrying the wild type RAS. g Representative IHC staining depicting the reduced p-ERK and p-AKT levels in 8305 C cell-derived xenograft mouse model, and quantitative IOD values shown in the right. Data shown as mean ± SD. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. SGSM2 inhibits the MAPK/ERK and PI3K/AKT networks via activation of RAP1.

a Lysates of 8305 C and FTC-133 overexpressing SGSM2 were immunoprecipitated with antibody against RAP1, and then immunoblotted with antibody targeting SGSM2 to confirm the intrinsic association between GMPS and TRIM21. b SGSM1 overexpression promoted RAP1 activation in indicated TC cells. Lysates of indicated cells were evaluated for RAP1 activation by detecting GTP-bound RAP1, and total RAP1 was determined using input samples. c Knock down RAP1 in K1, 8305 C, and FTC-133 cells restored p-ERK and p-AKT levels. d SGSM1 overexpression inhibited RAS activation in indicated TC cells. Lysates of indicated cells were employed for RAS activation assessment via evaluation of GTP-bound RAS levels, and total RAS levels were determined with input samples.

Fig. 5. A schematic model of SGSM2 suppressing the progression of RAS^WT thyroid cancer (TC).

SGSM2 binds and activates RAP1, and competitively inhibits the activities of wild type RAS. This diminishes the MAPK/ERK and PI3K/AKT networks in TC cells.