Abstract
Background/Aim: SPARC-related modular calcium-binding protein 2 (SMOC2), a secreted matricellular protein, is reported to be involved in cancer progression such as cell cycle, angiogenesis, and invasion. In this study, we aimed to investigate the expression of SMOC2 in various gastric lesions and assessed its prognostic value in a large cohort of gastric cancer (GC) patients. Patients and Methods: SMOC2 mRNA levels were measured by quantitative real-time PCR using 26 matched fresh-frozen GC samples. SMOC2 protein expression was determined by immunohistochemistry on tissue microarrays including 734 GC specimens and its correlations with clinicopathological features and survival were evaluated. Results: The transcription level of SMOC2 was higher in GC samples compared to normal mucosa (p=0.006). Its expression levels were associated with the intestinal stem cell (ISC) marker, LGR5, but there were no correlations with EPHB2 and OLFM4 or the candidate cancer stem cell markers CD133 and CD44. SMOC2 expression was significantly increased in the intestinal metaplasia and was further increased in gastric adenomas and early gastric cancers (EGC). In total, 34% of GCs were positive for SMOC2, and SMOC2 positivity was higher in old (p=0.001) and male (p<0.001) patients, and in well-differentiated GC (p<0.001). SMOC2 expression had a negative association with perineural invasion (p<0.001) and tumor stage (p<0.001). In survival analysis, SMOC2-positive GC patients had much better clinical outcomes in overall survival rates (p<0.001) compared to SMOC2-negative GC patients. The prognostic impact of SMOC2 remained significant both in intestinal (p<0.001) and diffuse-type GC (p<0.001). Remarkably, a multivariate analysis demonstrated SMOC2 as an independent prognostic marker [hazard ratio (HR)=0.732, p=0.045] along with venous invasion (p=0.012), tumor stage (p<0.001) and CDX2 (p=0.028). Conclusion: Our results suggest that SMOC2 can be a prognostic marker for better clinical outcomes in GC.
- SMOC2
- intestinal stem cell marker
- gastric cancer
- prognosis
- immunohistochemistry
Secreted protein acidic and rich in cysteine (SPARC)-related modular calcium-binding protein-2 (SMOC2), a secreted matricellular protein, is comprised of thyroglobulin type-I domains, EF-hand calcium-binding domains and a follistatin-like domain, and is widely expressed in many tissues (1). There have been studies investigating the implication of SMOC2 in a variety of human pathologies. In particular, SMOC2 is involved in various processes related to cancer progression, including cell proliferation (2), cell attachment and migration (3), angiogenesis (4) and fibrosis (5, 6). SMOC2 expression and its functional significance have also been explored in several types of malignancies such as colon (7-9), breast (10), liver (11, 12), lung (13), endometrium (14), pancreas (15) and thyroid cancers (16), yet the results remain controversial on whether it exerts an oncogenic or tumor-suppressive role in cancer development.
The significance of SMOC2 has been actively investigated in colorectal cancers (CRC) as it was identified to be one of the intestinal stem cell (ISC) markers (17). For instance, SMOC2 was shown to be necessary for more aggressive CRC properties and to act as a bone morphogenic protein (BMP) inhibitor in the intestine to enhance adenoma formation in Apc mutant mice (18). Most recently, SMOC2 was reported to be an independent prognostic marker for improved clinical outcomes in a large cohort of patients with CRC (8). However, there is no study so far reporting on SMOC2 expression in gastric cancers (GC) and its prognostic impact. Here, we aimed to investigate the expression profile of SMOC2 in various gastric pathologies, including a large cohort of GC, and evaluate its prognostic significance as well as its correlation with clinicopathological parameters.
Patients and Methods
Participants and tissue samples. This retrospective study enrolled GC samples (n=734) that were obtained from the patients who underwent curative gastrectomy at Seoul National University Hospital (SNUH) (Seoul, Korea) from 2004 to 2005. In addition, formalin-fixed and paraffin-embedded (FFPE) gastric lesions including normal gastric mucosa, intestinal metaplasia and gastric tumor specimens (n=159) were collected from the patients who underwent endoscopic submucosal dissection at SUNH from 2008 to 2010. Clinicopathological data included age, gender, tumor location, Lauren classification, histological type, presence of lympho-vascular and perineural invasion, pathological stage based on American Joint Committee on Cancer/International Union against Cancer (AJCC/UICC) cancer stage (8th edition). The histological tumor type and differentiation was classified according to the World Health Organization (WHO) criteria. Twenty-six unfixed, freshly-frozen GC tissues and matched noncancerous gastric tissues obtained from GC patients from 2001 to 2005 were provided by the Biobank of SNUH. For a validation study, a total of 875 GC patients were included from the data bases of Kaplan-Meier-plotter (http://kmplot.com/analysis). This study was approved by the Institutional Review Board of SNUH (H-1209-037-424) and JNUH (2017-06-029) and all procedures were in accordance with the ethical standards of the Helsinki Declaration of 1964 and later versions. The Institutional Review Board confirmed that informed consent was waived because of the retrospective nature of the study.
Tissue microarray (TMA) construction. In total, 14 TMAs containing 840 GC from gastrectomy specimens were assembled as previously described (19). Through light microscopy examination, a representative tumor area comprising more than 70% of tumor cell population was identified and marked on a hematoxylin and eosin (H&E) slide. Core tissue cylinders with a 2 mm in diameter were punched from each individual FFPE GC specimen of the paraffin block and arranged in a new recipient paraffin block using a trephine apparatus (SuperBioChips Laboratories, Seoul, Republic of Korea). Additionally, 3 TMAs were constructed, each of which contained 53 gastric tumors that had been removed by endoscopic submucosal dissection (ESD), and normal non-tumorous gastric mucosa samples, including the antral glands, fundic glands and intestinal metaplasia.
Immunohistochemistry. Immunohistochemistry (IHC) for SMOC2, CDX2 and β-catenin were performed on 4-μm TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s guidelines. The primary antibodies used were anti-SMOC2 (OriGene, Rockville, MD, USA; 1:30) anti-CDX2 (BioGenex, San Ramon, CA, USA; 1:500) and anti-β-catenin (Novocastra Laboratories, Newcastle, UK; 1:800). The expression of SMOC2 was determined by evaluating the cytoplasmic staining of tumor cells. The intensity of staining and percentage of tumor cells expressing SMOC2 were examined. Histoscores (H-scores) of SMOC2 were measured by multiplying the intensity score (0=negative; 1=weak; 2=moderate; 3=strong) and percentage of tumor cells expressing SMOC2 (range=0-100), and ranged from 0 to 300. For statistical analyses, we set a cutoff of 40 on the basis of the distribution of H-scores (average value: 32.5). SMOC2 positivity was determined as follows; GC with H-score <40 are negative, GC with H-score >40 are positive. CDX2 and β-catenin staining was defined as positive when more than 20% of the tumor cell nuclei were stained.
RNA extraction and quantitative real-time PCR. RNA extraction and real-time PCR was performed as previously described (19). In brief, total RNA was purified from 26 paired fresh GC samples and matched non-cancerous tissues using TRIZOL (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 1 to 2 μg of RNA using the GoScript reverse transcription system (Promega, Madison, WI, USA). Differential RNA levels were determined by performing real-time PCR with Premix EX Taq (Takara Bio, Shiga, Japan) as follows: initial denaturation for 30 s at 95°C, followed by 40 cycles of 95°C for 1 s and 60°C for 5 s in a StepOne Plus real-time PCR system (Applied Biosystems, USA). The TaqMan gene expression assays used were as follows: Hs00405777_m1 (SMOC2), Hs00173664_m1 (LGR5), Hs00197437 (OLFM4), Hs00362096-m1 (EPHB2), Hs01075864_m1 (CD44), Hs01009250_m1 (PROM1/CD133), and Hs0275899_g1 (GAPDH). GAPDH served as the endogenous control.
Statistical analysis. Statistical analyses were performed using SPSS software version 18.0 (SPSS, Chicago, IL, USA) and Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Between-group comparisons of the real-time PCR data were performed using Student’s t-test. The correlations between SMOC2 and intestinal stem cell markers were evaluated by Spearman correlation test. ANOVA with Bonferroni correction was used to compare H-scores of SMOC2 between various gastric lesions. Fisher’s exact test and Pearson chi-square test were used to assess statistical significance between SMOC2 positivity and clinicopathological parameters. Overall survival curves were estimated using Kaplan-Meier method and log-rank test was used to compare groups. Using Cox proportional hazards model, multivariate analyses were performed to calculate hazard ratios and 95% confidence intervals. A p-Value<0.05 was considered statistically significant.
Results
SMOC2 expression in gastric cancers and its correlation with stem cell-related markers. To determine the expression of SMOC2 and stem cell-related markers in GC, real-time PCR was performed on a series of 26 pairs of fresh GC samples as well as on matched normal gastric tissues. SMOC2 mRNA levels were higher in 58% of GCs (15 out of 26 cases) compared to normal mucosa in most samples (Figure 1A). The mean SMOC2 expression level was significantly higher in GC than in matched normal tissues (p=0.006) (Figure 1B). As SMOC2 is enriched in the stem cells of the intestinal crypts, we examined whether there was any correlation between SMOC2 and other ISC markers, including LGR5, EPHB2, and OLFM4 (Figure 1C-E). We found that only LGR5 demonstrated a positive association with SMOC2 (p=0.01). In addition, since a stem cell marker in the normal crypts is considered a promising cancer stem cell (CSC) marker, we also investigated the association of SMOC2 with candidate CSC markers in human GC; CD133 (Figure 1F) and CD44 (Figure 1G). Although SMOC2-high GC showed slightly higher CD133 expression levels, it did not reach statistical significance.
SMOC2 expression in normal mucosa and gastric tumors. IHC was performed in 3 tissue arrays, including normal gastric mucosa (antrum; n=7, corpus; n=3), intestinal metaplasia (IM) (n=17), benign (tubular adenoma with low-grade dysplasia; n==44, tubular adenoma with high-grade dysplasia; n=19) and malignant gastric tumors (n=62), to investigate the expression profile of SMOC2 during gastric cancer development. Immunohistochemical analysis on normal antrum only showed some SMOC2-positive cells at the gastric neck areas (Figure 2A). In the gastric corpus, SMOC2 staining was not observed except for non-specific cytoplasmic staining in chief cells (Figure 2B). Notably, SMOC2 expression was higher in IM (Figure 2C) and further increased in gastric adenomas (Figure 2D, E). Although SMOC2 levels in early GC were higher compared to normal antrum or corpus (Figure 2F), they were lower than those in tubular adenomas with high-grade dysplasia (Figure 2G). Overall H-scores of SMOC2 in various gastric lesions are presented in Figure 2G.
Associations of SMOC2 expression with clinicopathological parameters in GC. Next, IHC for SMOC2 was performed on 14 tissue arrays. GCs with H-scores of more than 40 were defined as positive for SMOC2. Representative images of SMOC2-negative and SMOC2-positive GC are presented in Figure 3A. The clinicopathological relevance of SMOC2 positivity is provided in Table I. SMOC2 positivity was higher in old (p=0.001) and male (p<0.001) GC patients, and in well-differentiated GC (p<0.001). SMOC2 expression was negatively associated with perineural invasion (p<0.001) and tumor stage (p<0.001), whereas no association was observed with tumor location (p=0.293), Lauren classification (p=0.969) lymphatic (p=0.066) and venous invasion (p=0.414). In addition, SMOC2 positivity had a positive association with CDX2 expression (p<0.001), while no association was detected with β-catenin expression (p=0.191).
Prognostic impact of SMOC2 in GC. Next, we evaluated the prognostic value of SMOC2 in a large cohort of GC patients (n=734). Representative images of SMOC2-negative and SMOC2-positive GCs are shown in Figure 3A. A survival analysis showed that SMOC2-positive GC patients had much better clinical outcomes in overall survival rates (p<0.001) than SMOC2-negative GC patients (Figure 3B). In addition, the prognostic impact of SMOC2 positivity remained significant both in the intestinal (p<0.001) and diffuse type (p<0.001) of GC (Figure 3B). When investigating the prognostic relevance of SMOC2 based on tumor-node-metastasis stages, it was only significant in stage III GC patients (Figure 3C). In a univariate analysis, several clinicopathological parameters were identified as prognostic markers, including age (HR=1.320, p=0.026), lymphatic (HR=3.845, p<0.001) and vascular invasion (HR=3.865, p=0.001), perineural invasion (HR=3.353, p<0.001), tumor stage (HR=3.953, p<0.001), CDX2 (HR=0.454, p<0.001) and SMOC2 (HR=0.473, p<0.001). Remarkably, a multivariate analysis revealed that SMOC2 was an independent prognostic marker (HR=0.732, p=0.045) along with venous invasion (HR=1.435, p=0.012), tumor stage (HR=3.411, p<0.001) and CDX2 (HR=0.628, p=0.028) (Table II). To verify the prognostic value of SMOC2, an independent analysis was performed with Kaplan-Meier-plotter for GC, a robust online tool enabling the validation of survival biomarker candidates based on transcriptome data (20). The Kaplan-Meier curves showed that high SMOC2 expression is significantly associated with lower survival rates (Figure 4), which is the opposite of what we observed in immunohistochemical analysis.
Discussion
In this study, we thoroughly examined the expression profile of SMOC2 in various gastric lesions, including a large cohort of GC, and evaluated its prognostic significance. First, we measured the SMOC2 mRNA level of GC samples and normal gastric tissue and found that SMOC2 expression was significantly higher in GC tissues than in normal gastric tissue. This finding is consistent with previous studies on colorectal cancer (CRC) (8) and hepatocellular carcinoma (HCC) (11, 12). In contrast, SMOC2 down-regulation was also reported in pancreatic (21), breast (22), and thyroid cancers (16). This discrepancy indicates that SMOC2 expression and its functional effect may differ in each cancer type. As SMOC2 is one of the enriched genes in stem cells of normal intestinal crypts, we investigated whether there was any correlation between SMOC2 and other well-known ISC markers, including LGR5, EPHB2, and OLFM4. We found that LGR5 demonstrated a positive association with SMOC2, while there were no correlations with EPHB2 and OLFM4 or candidate cancer stem cell markers. It could be worthy to further explore interactions between SMOC2 and LGR5 in GC.
Immunohistochemical analysis showed that some SMOC2-positive cells were observed in the neck region of the antral glands, while no SMOC2 expression was detected in the fundic glands. Interestingly, SMOC2 expression significantly increased in IM. IM is considered a precursor of gastric cancer and is thought to originate from isthmal stem or progenitor cells (23, 24). Previously, it has been shown that IM had a significantly higher rate of LGR5-positive cells (25) and those LGR5-positive cells in IM coexpress multiple ISC markers, indicating that an ISC population replaces the preexisting gastric stem cells (26). Therefore, given that SMOC2 belongs to ISC markers, it is not surprising to observe increased SMOC2 expression in an ISC population established in gastric IM. This close relationship of SMOC2 with IM may explain the strong positive correlation we observed between SMOC2 and CDX2 in GC, a transcription factor for intestinal differentiation.
When comparing SMOC2 levels between high-grade tubular adenomas and EGC, we saw that SMOC2 expression significantly declined in EGC. Furthermore, SMOC2 positivity was higher in well-differentiated and lower stage GC (Table I). This is consistent with recent results in CRC, in which SMOC2 expression was significantly reduced in the cancer cells at invasive fronts than in superficial areas (8). Likewise, in HCC, the SMOC2 expression level was higher in HCCs with smaller tumor size (<5 cm) and lower TNM stages (I-II) (12). This pattern of expression profile has also been reported with other ISC-related markers such as LGR5 (27) and EPHB2 (28) in CRC. These findings may suggest that SMOC2 is elevated in the early stages of tumor development and declines as cancer progresses into more advanced stages. Further studies are required to unveil the precise molecular mechanism of SMOC2 downregulation in advanced cancers.
We discovered that SMOC2 expression is associated with better clinical outcomes, particularly for stage III GC patients. However, we observed the opposite results in the independent survival analysis using Kaplan-Meier plotter, in which high SMOC2 expression was significantly associated with lower survival. This discrepancy may be primarily due to the difference in the method used to determine SMOC2 expression; we applied immunohistochemistry to detect SMOC2 protein and used H-scores to decide on SMOC2 positivity, whereas Kaplan-Meier plotter used the mRNA expression of SMOC2 from DNA chip data and each case was determined to be low or high based on the automatically selected cut-off value. In univariate and multivariate analyses, SMOC2 was an independent prognostic marker along with venous invasion, tumor stage and CDX2 (Table II). Our results of the SMOC2 expression profile in GC and its association with improved clinical outcomes suggest that SMOC2 may have tumor-suppressive roles in GC. This is in line with previous studies in CRC (8), thyroid cancer (16) and HCC (12). On the other hand, oncogenic properties of SMOC2 have been reported in other cancer types. For example, SMOC2 was associated with poor prognosis in endometrial cancer, and silencing SMOC2 reduced the clonogenic potential of endometrial cancer cells, while it downregulated the expression of the stemness-associated genes (14). In CRC, SMOC2 over-expression suppressed proliferation and migration, as well as colony and sphere-forming abilities (7). In breast cancer, knockdown of SMOC2 expression reduced the ability of the activated Ran mutant to stimulate anchorage-independent growth (10), and SMOC2 was required for clonal growth and efficient metastatic seeding in lung cancer (13). These findings suggest that SMOC2 may have dual oncogenic or tumor-suppressive roles in cancer progression depending on the cancer type.
In summary, SMOC2 expression levels were significantly higher in IM, gastric adenomas and GC, compared to corresponding normal tissues. Enhanced SMOC2 expression in tubular adenomas slightly declined in EGC. Remarkably, SMOC2 positivity was associated with prolonged survival rates and was shown to be an independent prognostic marker for better clinical outcomes. Taken together, the present study suggests that SMOC2 might be involved in the development of GC and thus may be a useful prognostic marker.
Acknowledgements
This work was supported by a research grant from Jeju National University Hospital in 2018.
Footnotes
# These Authors contributed equally to this work.
Authors’ Contributions
Chang Lim Hyun: Funding acquisition, Investigation. Sung Joon Park: Investigation, Writing-Original Draft. Hye Sung Kim, Hyun Joo Song, Heung Up Kim: Resources, Data curation. Young Hee Maeng, Young Sil Kim: Resources. Cheol Lee: Data curation. Dong Hui Lee: Software. Bogun Jang: Conceptualization, Supervision, Writing- Review & Editing.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received May 11, 2021.
- Revision received May 26, 2021.
- Accepted May 27, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.