As the CSC model has evolved, many biomarkers have been identified in various tumors to better understand CSCs and elucidate their roles in conferring stemness. SOX2, NANOG, and OCT4, contributing to the “core pluripotency network,” are transcription factors that regulate the development of embryonic stem cells (ESCs). These transcription factors are thought to regulate pluripotency and lead to self-renewal in embryonic and induced pluripotent stem cells [25, 26]. Moreover, it has been reported that SOX2 and OCT4 play roles in inducing the stemness of various cancer cells, as well as embryonic cells [26–28]. However, due to its rarity and a lack of understanding of its pathogenesis, only a few studies have been conducted on the clinical value of stem cell markers in small intestinal adenocarcinoma. Here, we investigated the clinical significance of SOX2, NANOG, and OCT4 expression in small intestinal adenocarcinomas.
We first determined SOX2 (35.1%), NANOG (50.8%), and OCT4 (44.3%) expression in small intestinal adenocarcinoma samples and demonstrated that SOX2 expression is an independent negative prognostic factor. Due to the lack of a standardized method for assessing SOX2 expression [13, 25], and the paucity of SOX2 nuclear expression, we used a histoscore method and digital image analysis to objectively quantify the results. In colorectal cancer samples, SOX2 expression has been analyzed with an absolute quantitative or semiquantitative scoring system via microscopy. It has been reported that SOX2 expression ranges from 11–45.6% and that it correlates with lymph node metastasis, tumor grade, TNM categories, and BRAF mutations [13, 25].
In this study, we observed that OCT4 is mostly expressed in the cytoplasm and is associated with low pT category and stage grouping. When considering different isoforms of OCT4, OCT4A is a nuclear transcription factor responsible for the pluripotency properties of ESCs, while OCT4B resides in the cytoplasm, where it may respond to cellular stress . Therefore, it has been suggested that OCT4B expression is predominantly found in small intestinal adenocarcinoma cells, which correlates with low tumor stage. Additionally, alternatively spliced OCT4 transcripts may exhibit diverse functions in different tissues, considering that OCT4 expression (mean, 12.4%) is also found in normal small intestinal mucosa.
There is ongoing debate about the contribution of the Wnt signaling pathway to self-renewal and differentiation in human ESCs; however, many members of the Wnt signaling pathway are implicated in stem-cell proliferation [26, 30]. Recently, Moon et al. demonstrated that the initial activation of β-catenin, by APC loss, and further enhancement through mutated KRAS induces CD44, CD133, and CD166 expression in colorectal cancer . Moreover, many studies suggest that SOX2, NANOG, and OCT4 expression may contribute to both the EMT and stemness of cancer cells in the digestive system [14, 15, 31]. Although small intestinal adenocarcinomas express notably different levels of APC (26.8% vs 75.9%), CDKN2A (14.5% vs 2.6%) and TP53 (58.4% vs 75.0%), they are characterized by alterations in both KRAS (53.6%) and PIK3CA (16.1%), which are involved in dedifferentiation and disease progression . Therefore, we analyzed the expression of SOX2, NANOG, and OCT4 in conjunction with the KRAS genotype in small intestinal adenocarcinoma patients. SOX2 expression was higher in the KRASMT subgroup (40.0%) than in the KRASWT (32.8%) subgroup, but this difference was not statistically significant. We found that patients with KRASMT and SOX2 expression had significantly worse OS outcomes than those with KRASWT without SOX2 expression. This implies that both SOX2 and KRAS genotypes are important prognostic factors for small intestinal adenocarcinomas. The KRASWT/SOX2+ expression pattern was more frequently found in association with high-grade carcinomas than with low-grade carcinomas, which suggests that SOX2 may play a certain role in the dedifferentiation of small intestinal adenocarcinoma cells, independently of KRAS genotype. Although there was no statistical significance with SOX2+ expression and tumor differentiation, we observed SOX2+ expression to be more common in high-grade tumors (44.4%, 20/45) than low-grade tumors (32.1%, 45/140) (Supplementary Table 2).
We found small intestinal adenocarcinomas with KRASMT and NANOG+ expression to be associated with a lack of lymphovascular tumor invasion and small intestinal adenocarcinomas with KRASMT and OCT4+expression to be associated with eary stage group. Regarding the combined expression of SOX2, NANOG, and OCT4, we observed that NANOG+/OCT4+ expression was more common in early-stage carcinoma, whereas SOX2+/OCT4+ and SOX2+/NANOG+/OCT4+ expression were associated with short OS. It is well known that the transcription factor NANOG is localized to the nucleus . We detected that NANOG is expressed in the cytoplasm of small intestinal cancer cells with and without nuclear accumulation, which corroborates the findings of other colorectal cancer studies [14, 33]. This aberrant expression pattern is frequently found in a variety of cancers, with testicular germ cell tumors being a notable exception . The regulation mechanism for localization is currently unknown and should be elucidated in the future. Recent studies also indicate that NANOG is a negative prognostic factor among colorectal cancer patients. Meng et al. revealed that NANOG expression significantly correlates with poor prognosis, lymph node metastasis, and TNM categories , and Xu et al. reported that NANOG may be a potential biomarker for the postoperative hepatic metastasis of colorectal cancer . These results suggest that SOX2, NANOG, and OCT4 play complex roles in small intestinal adenocarcinoma. Further studies are needed to clarify the interaction between SOX2, NANOG, OCT4, and KRAS mutations in small intestinal adenocarcinoma.
The main challenge of this study was that it relied on a relatively imbalanced cohort, despite collecting patient samples from multiple institutions. In this study cohort, 57% of the patients had pT4 tumors, and 52% had cancers with AJCC stage group III, even though inoperable stage IV cases with distant metastases were not included. However, this deviation seems to be characteristic of small intestinal adenocarcinoma. Indeed, the findings of a previous large single-center study agreed with our findings when comparing just the percentages of stage I, II, and III cases, which were 12%, 45%, and 43%, respectively. Additionally, other epidemiologic characteristics, such as age, sex, and location, also paralleled those of our cohort .