Role of RUNX2 in Oral Squamous Cell Carcinoma (OSCC): A Systematic Scoping Review

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Introduction
RUNX2, known as core binding factor alpha 1subunit (Cbfa1), Pebp2αA is a member of the RUNX family of transcription factors, which are characterized by their DNAbinding domains, known as the Runt domains [1].RUNX2 is a transcription factor protein that is essential for bone formation and is known to play an important role in various other biological processes [2].
In bone formation, RUNX2 is a master regulator of osteoblast diferentiation and bone formation.It has been shown to be essential for the responsibility of mesenchymal stem cells to the osteoblast lineage, as well as for the differentiation of preosteoblasts into mature osteoblasts [3].RUNX2 is also involved in the mineralization of bone matrix and the regulation of bone remodeling (Figure 1) [3,4].RUNX2 is also involved in other biological processes, including the regulation of tooth development, chondrogenesis, and the maintenance of the hematopoietic stem cell [5,6].In addition, it has been shown to play a role in the regulation of angiogenesis and tumor metastasis [7].
Osteoblasts produce and mineralize the extracellular matrix of bone, essential for maintaining skeletal integrity.RUNX2 activates the expression of various genes involved in osteoblast diferentiation, including alkaline phosphatase (ALP), osteocalcin (OCN), and collagen type I (COL1A1) [8].Furthermore, RUNX2 is also involved in maintaining bone homeostasis and remodeling by regulating osteoclast diferentiation and activity [9].RUNX2 is also implicated in chondrogenesis, the process by which cartilage is formed.In chondrocytes, RUNX2 plays a role in regulating the expression of genes involved in chondrocyte diferentiation, such as type II collagen (COL2A1) and aggrecan (ACAN) [10,11].Additionally, RUNX2 is involved in developing craniofacial bones and teeth [12].
RUNX2 is a critical factor in bone formation and other biological processes, and its dysregulation has been implicated in a variety of skeletal and nonskeletal diseases [13].For example, mutations in the RUNX2 gene have been associated with cleidocranial dysplasia, a skeletal disorder characterized by abnormal bone development [14].RUNX2 has also been implicated in developing and progressing breast and prostate cancer [15,16].Te function of RUNX2 is regulated by a variety of signaling pathways, including the Wnt, BMP, FGF, Shh, and PTH pathways [4], and RUNX2 activity is also modulated by posttranslational modifcations, including phosphorylation, acetylation, and SUMOylation [17].Runx2-dependent transcription is regulated by posttranslational modifcations and interactions with additional nuclear factors.Te key components involved in these modifcations include chromatin remodelers such as the Swi/Snf complex [18], ATPase proteins Rvb1 and Rvb2 [19], and histone 3 lysine 9 (H3K9) demethylases like JMJD2d/ Kdm4d [20].Te Swi/Snf complex is required for cotranscriptional nucleosome remodeling, leading to transcriptional interference and repression of gene expression [21].Rvb1 and Rvb2 proteins are enriched at gene promoters and mRNAs, where they sequester mRNAs into mRNP granules and repress their translation [22].JMJD2d/Kdm4d demethylase regulates type I interferon responses by afecting the transcription of enhancer RNAs (eRNAs) and dynamic H3K9me2 at associated promoters.Tese components play crucial roles in regulating Runx2-dependent transcription through various mechanisms, including chromatin remodeling, mRNA localization, and translatability.Te main driving components that play a major role in dysregulation and aberrant expression of RUNX2 in cancer are several.One mechanism involves the cooperation of enhancers and chromatin looping, which is controlled by the transcription factor c-JUN and the epigenetic regulator BRD4 [23].Another mechanism involves the activity of histone deacetylases (HDACs), particularly HDAC1 and HDAC6, which are required for efcient transcription of RUNX2 in cancer cells [24].Additionally, constitutive activation of signaling pathways, such as ERK, Smads, cdks, and Akt, can lead to aberrant expression and activation of RUNX2 [24].Treatment with histone deacetylase inhibitors (HDACi) can inhibit RUNX2 expression by disrupting the transcription-activating complex on the enhancer element within the RUNX2 gene [25].Tese fndings suggest that dysregulation and aberrant expression of RUNX2 in cancer can result from a combination of enhancer-mediated chromatin looping, epigenetic regulation by HDACs, and activation of signaling pathways.Recent studies have suggested that RUNX2 may also have a role in cancer.In particular, RUNX2 has been found to be overexpressed in various types of cancer, including breast, prostate, and lung cancer [15,26,27].RUNX2 is thought to promote tumor growth and metastasis by inducing the expression of genes involved in angiogenesis, cell migration, and invasion.A study by Yang DP et al. (2020) found RUNX2 was overexpressed in lung squamous carcinoma and correlated with the clinical progression of this fatal disease [27].
Dysregulation and aberrant expression of RUNX2 have been linked to the pathogenesis of OSCC.Its dysregulation promotes tumor initiation, progression, and metastasis [28].Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, and OSCC is the most common malignant type of HNSCC [29,30].Cancer is a leading cause of death globally, accounting for one in six deaths or nearly 10 million in 2020 [31].With recent scientifc advances in cancer detection and treatment, the survival rate of many cancer types has witnessed tremendous improvement.However, HNSCC has a poor survival rate with the 5year survival rate at an abysmally low level of 50%.Recurrence and metastasis are the main reasons for the poor survival rate [32].Recurrence occurs in about half of all HNSCC patients [33].Hence, novel therapeutic strategies aimed at controlling recurrence and metastasis can go a long way in improving the prognosis and survival rate of the patients.
Tis review systematically identifed evidence from the recent research studies on the role of RUNX2 in HNSCC.It explored the possibility that RUNX2 could be used as a biomarker for the prognosis of OSCC.Te prevailing perception and scope of development of therapeutic strategies using RUNX2 to treat OSCC are emphasized.

Methodology
Before the advent of the current review, a preliminary search of MEDLINE, the Web of Science, and JBI Evidence Synthesis was conducted, and no current or ongoing systematic reviews or scoping reviews on the topic were identifed.Te current scoping review was conducted following the framework designed by Arksey and O'Malley following the 5-step framework [34]. 2

Identifying the Research Question
European Journal of Cancer Care subquestions were "What are the types of studies conducted on the role of RUNX2 in OSCC?" "Does RUNX2 have any prognostic or therapeutic role in OSCC?" and "What are the gaps in the literature available on this topic?".(ii) Identifying relevant studies Following the PCC (population, concept, and context) criteria, with the population being OSCC and HNSCC patients and the concept being the role of RUNX2, relevant studies were identifed using specifc eligibility criteria.Only original research articles published in peer-reviewed journals and in English language were considered for the review.Preprints were excluded.An elaborate scientifc literature search was executed with no start date restriction until 8 September 2023 in the electronic databases MEDLINE, Web of Science, and Scopus, following specifc eligibility criteria.Te protocol for search terms and the search strategy used for MEDLINE and Web of Science is given in Tables 1-3, respectively.

Selection of Studies for Inclusion.
Te results of the search and the study inclusion process are presented in a Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping review (PRISMA-ScR) fow diagram (Figure 2) [35].After reading through the title and abstract of the articles, one article was excluded for being a meeting abstract, whose full text was not available online.After full-text screening was performed, two articles were excluded because they were not appropriate for the review.Finally, seven articles were included in this review.Following the search, all identifed citations were collated and uploaded into EndNote version 21, and duplicates were removed.Titles and abstracts were screened for assessment against the inclusion criteria for the review.A total of 10 studies were selected by this method.

Data Extraction and Charting of Data.
After a thorough reading of the selected articles, the required data were extracted and entered into MS word.Te collected data included authors, date of publication, study type, study population, and key points to be taken from the study (Table 3).Te data were regularly updated and meticulously charted.

Collating, Summarizing, and Reporting of Results.
A thorough summary of the results was prepared, and a narrative approach is used to report the results of this systematic scoping review using fgures and tables [34,36].

Results and Discussion
Te result of the review is given in Table 3. Te role of RUNX2 in OSCC was thoroughly studied and summarized legibly in this review.
3.1.RUNX2 and OSCC.High RUNX2 expression levels have been observed in OSCC tissues and are associated with tumor growth, invasion, and metastasis.RUNX2 interacts with several signaling pathways involved in OSCC pathogenesis, such as the Hedgehog signaling pathway.Zhang et al. mentioned that RUNX2 promoted fbroblast activation and OSCC cell proliferation and migration [28].It is a multipotent transcription factor that regulates various genes and interacts with diferent oncogenic transcription factors of HNSCC, like TP53, JUN, and HIF-1A [37][38][39][40].
RUNX2 plays a major role in tumor cells' transformation, growth, metastasis, cachexia, and hypercalcemia [7,41].RUNX2 crosstalks with other transcription factors to regulate the function of oncogenes and cancer progression.It also has a prometastatic function, playing a signifcant role in the cervical metastasis in HNSCC [23].In HNSCC, RUNX2 collaborates or engages in crosstalk with other transcription factors to regulate oncogenes' functions and infuence the progression of cancer [23].Chang et al. found that in HNSCC, RUNX2 could have a specifc transcription target and demonstrated the prometastatic function of RUNX2 [23].
Increased expression of RUNX2 following the knockdown of tumor suppressor microRNA, miRNA-376c-3p, leads to an upregulation of invasive and migratory abilities of tumor cells and increased infltration of cervical lymph nodes [23] .

RUNX2 as a Prognostic Biomarker. RUNX2 has been linked to the development of metastasis in various cancers.
In OSCC, its expression may be associated with an increased risk of lymph node metastasis [23].Terefore, assessing RUNX2 levels could aid in predicting the likelihood of metastasis and tailoring treatment strategies accordingly.
RUNX2 mRNA and protein levels were found to be elevated in clinical HNSCC cases [45].A study by Chang et al. showed RUNX2 to stimulate HNSCC via parathyroid hormone-like hormone (PTHLH) and was found to be a poor prognostic marker for HNSCC [45].It was found that PTHLH, calcium, and RUNX2 form a positive feedback loop in HNSCC, and in HNSCC patients, the RUNX2-PTHLH axis promoted tumor growth [45].Recent studies show that the most activated upstream regulator in highly metastatic HNSCC was RUNX2 [23,49].RUNX2 was found to promote the proliferation abilities of HNSCC cells in vitro and tumor growth in vivo [45].
MRE11 is crucial for DNA double-strand breaks and is the nuclease component of the RAD50/MRE11/NBS1 DNA repair complex.It is also found to be a critical factor in cancer development [50].In OSCC, MRE11 expression was associated with increased tumor size, cancer stage, and lymph node metastasis.It also predicted poorer patient survival and radiotherapy resistance [43].Wang et al. found that MRE11 enhanced tumor proliferation, migration, and invasion, which was mediated by RUNX2 CXCR4, AKT, and FOXA2 [43].

Targeting RUNX2 for OSCC Terapy.
MicroRNAs have several translational uses, including being diagnostic and prognostic biomarkers.MicroRNA miR-23a was found to inhibit invasion, proliferation, and clonogenesis of OSCC cells by inhibiting RUNX2 regulation of the PTEN/PI3K/Akt signaling pathway [42].Chang et al. suggested that therapeutic targeting of the transcriptional activity of RUNX2 by  European Journal of Cancer Care RUNX2's involvement in the progression of oral cancer makes it a potential therapeutic target.Inhibiting or modulating RUNX2 activity could be explored to suppress tumor growth and metastasis.Targeting RUNX2 may represent a promising therapeutic approach for OSCC treatment.Studies have shown that RUNX2 inhibition using siRNA or small molecule inhibitors suppresses OSCC cell growth, invasion, and metastasis in vitro and in vivo [23].Moreover, RUNX2 inhibition has sensitized OSCC cells to chemotherapy and radiation therapy.However, more studies are needed to determine the safety and efcacy of RUNX2 inhibition as a therapeutic approach for OSCC.

Conclusion
RUNX2 is a critical transcription factor that plays a key role in bone formation and other biological processes.Various signaling pathways and posttranslational modifcations tightly regulate its activity.Dysregulation of RUNX2 has been implicated in various skeletal and nonskeletal diseases.Recent studies have implicated RUNX2 in cancer, suggesting a potential therapeutic target for the treatment of cancer, including OSCC.Tey may serve as a prognostic biomarker and therapeutic target for OSCC treatment.
Elevated levels of RUNX2 expression have been detected in OSCC tissues and are correlated with the growth, invasion, and metastasis of tumors.RUNX2 has been associated with the development of metastasis across various cancers, and in the case of OSCC, its expression may be linked to an increased risk of lymph node metastasis.Consequently, evaluating the levels of RUNX2 could assist in predicting the likelihood of metastasis, guiding treatment strategies accordingly.Moreover, heightened RUNX2 expression has been associated with an unfavorable prognosis in OSCC patients, suggesting the potential utility of RUNX2 as a prognostic biomarker.Given RUNX2's role in the progression of oral cancer, it emerges as a plausible therapeutic target.Exploring the inhibition or modulation of RUNX2 activity could be considered to suppress both tumor growth and metastasis.Terefore, targeting RUNX2 presents a promising therapeutic approach for the treatment of OSCC.Tere is a dearth of studies analyzing the molecular mechanisms involved in the role of RUNX2 in OSCC.Future studies should further elucidate the molecular mechanisms underlying RUNX2-mediated OSCC pathogenesis and develop safe and efective RUNX2-targeted therapies for OSCC patients.

Figure 1 :
Figure 1: Summarize RUNX2 role of regulation and diferentiation in mesenchymal stem cells for osteoblast cells and contribution to chondrocyte maturation [40].

Figure 2 :
Figure 2: PRISMA-ScR fow diagram for studies included in the review.

Table 2 :
MEDLINE search strategy.

Table 3 :
Web of Science search strategy.

Table 4 :
List of studies included.