Expression profile of cancer stem cell markers SOX2, OCT4 & NANOG in salivary gland malignancies: A systematic review

Materials & Methods

The protocol of this systematic review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) database (CRD42023408336) and conducted in agreement with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines.

Research question

This systematic review addressed the following research question: Do SOX2, OCT4, and NANOGSCMs determine the behaviour and prognosis of salivary gland malignancies? The Population Intervention Comparison Outcome (PICO) used to define the research question was:

Results

Study selection

Out of the 218 articles identified from the databases, 126 duplicate studies were removed. Further, 92 articles were screened, and 31 were assessed for eligibility. After considering the desired inclusion criteria and identifying articles from other sources, 10 studies that met the eligibility criteria were included (Fig. 1).

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Fig. 1.

Flowchart depicting Identification, screening and inclusion of the article.

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Quality assessment

All the studies were of high quality, with scores 7 and 9 in six and four studies, respectively (Fig. 2 and 3).

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Fig. 2.

Risk of bias assessed by the Newcastle-Ottawa quality assessment scale for case-control studies.

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Fig. 3.

Risk of bias assessed by the modified Newcastle-Ottawa quality assessment scale for a cross-sectional study.

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Discussion

Cancer seeding and advancement are governed by a small subpopulation of self-renewing and differentiating oncogenic CSCs²⁰. In salivary gland malignancies, characterisation of CSCs could provide a better insight into the pathobiology and aid in the development of more effective therapies. Thus, currently, squamous cell metaplasias (SCMs) are being investigated to identify CSCs, and their expression profile is correlated with the clinicopathological features²¹. Molecular and immunohistochemical markers are a boon for CSC research as they help to identify and isolate CSCs and explore the pathways of oncogenesis, thus, establishing a novel target for therapeutic purposes¹¹.

The core transcription factors SOX-2, OCT4, and NANOG play an imperative role in the retention of the pluripotent ESC phenotype through self-controlling, feedback, and feed forward regulatory circuits²². These ESC biomarkers are reported to have a strong correlation with CSCs as both share specific tumour characteristics, and certain mutual molecular cross-talks might exist between the two celltypes¹⁹. Biomarkers SOX-2, OCT4, and NANOG independently and synergistically interact bringing transcriptional alteration in the genetic pathways. SOX2 and OCT4 have a common DNA-binding site, thus regulating the target genes by forming heterodimers. In addition, SOX2 keeps OCT4 expression at suitable levels²³. Moreover, NANOG is an established target modulated by OCT4/SOX2; thus, a well-linked transcriptional control looped pathway has been recognised²⁴. Furthermore, these proteins have been substantially related to EMT and various clinicopathological factors². The early transcription factors can mediate tumour initiation, proliferation, differentiation, metastasis, relapse, and therefore, increased expression of studied proteins has been linked with worse prognosis and dismal outcomes in various human malignancies²⁵. The elucidation of the molecular intricacies causing varied expression of CSC markers in salivary gland malignancies through additional clinical or experimental studies might help to substantiate these transcription factors as unique potential targets for predictive and therapeutic approaches²⁶.

In salivary glands, the intercalated and excretory ducts are the locus for proliferating cells, and acinar components have a limited degree of proliferative ability, implying that a population of potent progenitor stem cells resides in the ducts¹⁰ SOX-2 protects CSCs against apoptosis and is significantly responsible for their longevity. Highly diverse expression patterns and correlation with clinicopathological outcome are suggestive of the distinctive role of SOX2 in individual tumours²⁷. In salivary gland carcinomas, higher SOX2 expression indicates increased CSC activity and, therefore, could be a reliable marker for predicting and may be a potential therapeutic target requiring more aggressive therapy²¹. Dai et al¹⁰ in ACC observed poorer overall and disease-free survival with higher expression of SOX2. Higher expression of CD44 and SOX2 in primary squamous cell carcinoma and high-grade MEC suggests that therapies combining these markers might benefit the patients¹⁶. In addition, Xu et al¹³ stated that the amalgamation of biomarkers SOX2/CD44/CD133 has prognostic significance in the MEC of the palate. However, Sadeghi et al²⁸ found a considerable link between higher grades of MEC and low SOX2 expression and poor outcomes in ACC patients. Luo et al¹⁶ reported significantly marked expression of SOX2 and surviving in ACC as compared to normal salivary gland tissues and stated that both markers are tissue-specific and may become salivary gland ACC oncogenes. They also observed increased SOX2 expression in the peripheral region of the tumour pointing towards invasive, aggressive, and malignant behaviour of the tumour. Bertlicha et al¹⁷ also found that elevated SOX-2 expression is associated with disease aggressiveness and a poorer prognosis. Thierauf et al¹⁴ postulated a context-dependent function and regulation of SOX2 in distinct malignancies.

Human adult stem cells, germ cells and ESCs express transcription factors OCT4 and NANOG with higher proliferative capacity leading to aggressive tumour biology²⁹. The potential of NANOG and OCT-4 to act as a prognostic biomarker has been explored by researchers in the malignancies of salivary glands and other head and neck regions³⁰. An association between the NANOG and OCT4 expression with perineural invasion, other histopathological features, and positive expression in ductal structures in MEC has been related to destructive behaviours and worst outcomes. In addition, tumour-initiating abilities and metastasis have been controlled by OCT4/NANOG signalling through EMT¹⁸. Higher OCT-4 expression in ACCs and MECs has been attributed to characteristics inherent to embryonic development through EMT²⁹. However, Spiegel et al¹⁹ observed lower NANOG and SOX2 expression without any noteworthy association with survival estimates and stated that both markers have no prognostic value in salivary gland adenocarcinoma. Santos et al³¹ found raised expression of OCT-4 and SOX2 in lesions without myoepithelial differentiation and malignant behaviour. However, stromal immunoexpression of OCT4 was higher in malignant salivary gland tumours and was related to myoepithelial differentiation.

The present systematic review provides evidence for the higher expression of pluripotent CSC markers in malignant salivary gland tumours and their significant association with higher-grade and advanced-stage malignancies, although contradictory findings about therapeutic effectiveness were present. Therapeutic modalities of salivary gland malignancies are particularly challenging because of disease rarity and heterogeneity¹. Poor prognosis and dismal median overall survival (OS) of 15 months and five-year OS rates of about 15 per cent have been seen in patients with recurrent or metastatic (R/M) disease³². Therefore, selectively targeting CSCs might hinder recurrence and metastasis of the tumour thereby improving outcome and patient’s quality of life¹⁶ . Experimental research has documented that induced pluripotent stem cells (iPS) could treat salivary gland tumours and restore glandular function³³. The rationalisation of therapeutic strategies tailored to the patient’s specific cancer type and prognosis of R/M disease can be significantly improved by unravelling immunohistochemical expression profiles and mapping genomic variations in salivary gland malignancies, thus justifying and warranting biomarker-driven research^34,35.

There were few limitations in this systematic review. The included studies in the present systematic review are retrospective and have used immunohistochemistry as a primary investigative tool, and paraffin-embedded tissues archived for longer periods could affect immunohistochemical expression of the markers. Most of the studies have smaller sample sizes because of the low incidence of certain salivary gland malignancies and the non-availability of clinical prospective trials. Studies used different therapeutic approaches that could be one of the confounding factors for clinical outcomes. The inability to conduct meta-analysis is one of the major limitations of the present systematic review because of the lack of relevant data about the expression profile of CSC markers with survival rates, grading, and clinical stages. In addition, the number of studies retrieved on specific salivary gland malignancies was less, making meta-analysis impossible. Another major constraint was the inability to explore heterogeneity due to a lack of relevant data. In addition, no access to some full-text articles, non-inclusion of studies in a language other than English, and some that failed to produce salivary gland malignancy-specific data could also result in publication bias.

Overall, in malignancies, substituting conventional treatments with more efficient options have been revolutionised by the emergence and detection of CSCs by specific biomarkers with a deep influence on the expansion of molecular therapeutic strategies. SOX2, OCT-4, and NANOG have been proven as prognosticators in salivary gland malignancies, with a definite role in tumour biology. The selective targeting and unravelling of tumorigenic CSCs, genetic alterations, and protein expression profiles of salivary gland malignancies could rationalise treatment modalities tailored to the patient’s tumour.