UCLA

Background: Head and neck cancer includes various tumors which originate in oral cavity, laryngeal, and pharyngeal sites. Globally, it is the sixth most common neoplasia with 6% patient cases diagnosed for this disease.[1] Histologically, more than 95% of head and neck cancer is classified as squamous cell carcinoma (SCC).[2] These are aggressive tumors with a mortality rate between 1-2%.[1] To increase patient survival, it is important to discover new relevant biomarkers for early detection, targeted therapies and screening for relapse or secondary lesions for improved cancer management.

Sex-determining region Y-gene (SRY) box 5 protein (SOX5), member of the subgroup D of SOX superfamily.[3] Recent literature in the cancer biology revealed that they are highly conserved transcription factors family and SOX5 has been involved in epithelial to mesenchymal transition (EMT) in cancers like breast cancer, hepatocellular cancer, prostate cancer, lung adenocarcinoma, osteosarcoma and nasopharyngeal cancer.[4-9] Our preliminary research has shown higher endogenous SOX5 expression in head and neck squamous cell carcinoma (HNSCC) cell lines. Also, SOX5 knockdown has a regressive effect on the cancer progression in HNSCC.

Our earlier study also demonstrated that among the growth factors only epithelial growth factor (EGF) treatment to the cancer cells evidently upregulated SOX5 expression. EGF and its receptor epidermal growth factor receptor (EGFR) are expressed at much higher levels in HNSCC than in normal epithelial tissue and correlate with poor prognosis.[10, 11] The downstream target effectors of EGFR include the signal transducers and activators of transcription-3 (STAT3) which is constitutively activated in HNSCC.[10-13] SOX5 has also been found as a downstream target of STAT3 in murine Th17 cells and in B-cell lymphoma.[14-16] Thus, investigating the EGF and EGFR signaling via STAT3 is very important to identify their role in SOX5 regulation.

TWIST-1 and SNAIL-1 are already well-established downstream targets of SOX5 aiding in the process of EMT in some cancers. TWIST-1 play key roles in embryonic development, while mostly undetectable in healthy adult tissues. It is frequently reactivated in a wide array of human cancers[17-20]. It is also correlated with more metastatic lesions in head and neck cancer.[17] However, it’s unclear about the mechanism of TWIST-1 activation and its upstream signaling pathway during tumorigenesis.

Objectives: This study aims to identify a mechanism of regulation of SOX5 in head and neck cancer progression. As higher expression of both EGF, EGFR and SOX5 separately correlated with poor clinical prognosis in cancer research. Also, SOX5 has been found as a downstream target of STAT3 in few cancers. Establishing a link between active form of STAT3, i.e. phosphorylated STAT3 (phospho-STAT3), and SOX5 will be crucial to illustrate a pathway of SOX5 activation and regulation. Moreover, in some cancers TWIST-1 and SNAIL-1 are found as downstream targets of SOX5 aiding in the process of EMT. It could be interesting to identify the downstream targets of SOX5 in HNSCC.

Methods: Western blotting and qPCR were used to quantify SOX5 levels and some EMT markers across SOX5 knockdown, EGF treatment and SOX5 overexpression in UM1 and UM5 cells as the pathways potentiating the invasiveness are prominent in these two highly invasive head and neck cancer cell lines. Phenotypic studies were performed with EGF treatment and SOX5 overexpression. MTT, migration, and invasion assays were utilized to assess phenotypes. Chromatin immunoprecipitation (ChIP) assay was used to examine the regulatory potential of phospho-STAT3 with SOX5 promoter and of SOX5 with TWIST-1 promoter. Luciferase assay was used to examine the regulation of TWIST-1 promoter by SOX5.

Results: With EGF treatment, UM1 and UM5 exhibited an increased ability to proliferate, migrate and invade. With higher levels of SOX5 after the overexpression of SOX5, there was an increase in proliferation, migration, and invasion potential in UM1 and UM5. ChIP assay results suggested binding of phospho-STAT3 to SOX5 promoter following STAT3 immunoprecipitation and qPCR analysis of the SOX5 promoter in UM1 and UM5. ChIP assay and Luciferase assay results indicated binding of SOX5 to TWIST-1 promoter.

Conclusions: This study observes the impact of transcription factor SOX5 in the progression of head and neck squamous cell carcinoma. SOX5 has been found to be critical in EMT causing progression of HNSCC, as it has been shown in other types of cancers. Both upregulation and knockdown of SOX5 have provided evidence to characterize its oncogenic effect in HNSCC cells. This study also found a few of the potential targets of action and regulation of SOX5. A link between EGF, STAT3, SOX5 and TWIST-1 in a regulatory pathway is established suggesting that STAT3 may regulate SOX5 which may further regulate TWIST-1. Recognizing this mechanism of SOX5 in head and neck cancer provides additional insightful knowledge about the role of SOX family in cancer. With more validating research, SOX5 could be used as a prognostic biomarker in HNSCC.