KLF4, Slug and EMT in Head and Neck Squamous Cell Carcinoma

Epithelial to mesenchymal transition (EMT) is clinically relevant in head and neck squamous cell carcinoma (HNSCC). We hypothesized that EMT-transcription factors (EMT-TFs) and an anti-EMT factor, Krüppel-like-factor-4 (KLF4) regulate EMT in HNSCC. Ten control mucosa and 37 HNSCC tissue samples and three HNSCC cell lines were included for investigation of EMT-TFs, KLF4 and vimentin at mRNA and protein levels. Slug gene expression was significantly higher, whereas, KLF4 gene expression was significantly lower in HNSCC than in normal mucosa. In the majority of HNSCC samples, there was a significant negative correlation between KLF4 and Slug gene expression. Slug gene expression was significantly higher in human papilloma virus (HPV) negative HNSCC, and in tumor samples with irregular p53 gene sequence. Transforming-growth-factor-beta-1 (TGF- β1) contributed to downregulation of KLF4 and upregulation of Slug. Two possible regulatory pathways could be suggested: (1) EMT-factors induced pathway, where TGF-β1 induced Slug together with vimentin, and KLF4 was down regulated at the same time; (2) p53 mutations contributed to upregulation and stabilization of Slug, where also KLF4 could co-exist with EMT-TFs.


Supplementary Material
Supplementary

Experiments to differentiate between Slug and Snail in HNSCC tissue samples
Interestingly, immunohistochemical reactions with Slug antibodies produced for the whole recombinant protein, as most of the commercial ones including the clone S43-1259 (BD Pharmingen), hardly distinguish between Snail and Slug proteins (Supplementary Figure 1). Even western blot does not allow a distinguish between Snail and Slug, because both proteins show a 30 kDa product. Only few amino acids, short peptide components allow a differentiation between Snail and Slug (Supplementary Figure 1), but antibodies even used in papers, which distinguish between Snail and Slug [25] are frequently withdrawn by providers. This is an important problem, while since the publication of Ye et al.
[7], it is highly important to distinguish between Snail and Slug.
In addition, the mouse monoclonal Snail antibody (clone G-7, Santa Cruz Biotechnology, Heidelberg, Germany), which recognizes the peptide sequence exclusive in Snail and not present in Slug protein (Supplementary Figure 1), does not show positive reaction in HNSCC tissue, which is intensive stained by Slug antibody (not shown).

Supplementary Figure S1
Commercial antibodies against Snail or Slug do not allow the distinguish between the two proteins If whole recombinant protein was used for producing SLUG antibody, as in the case of commercial antibodies, the following coverage is found for SNAI1 gene product: SNAIL protein: As seen here, there is a high range overlap between snail and slug at protein level.
The peptide sequence of SNAIL amino acids 113-139 is not present in Slug.

FSSTSVSSLEAEAYAAFPGLGQVPKQL
The antibody produced against Snail amino acids 113-139 has full reactivity with Snail; BUT NO REACTION WITH SLUG AT ALL.
This antibody is available for Santa Cruz Biotech and can be found by the following link: https://datasheets.scbt.com/sc-271977.pdf In our material, tissue samples showing high reaction with Slug antibodies do not react with this Santa Cruz Biotech antibody.

Supplementary Technical Supporting Data-2
Gene expression analysis of SNAI1, SLUG, ZEB1, TWIST and KLF4 in HNSCC compared with normal oral mucosa The CT-values of SNAI1 ranged from 34 to 38 both in control normal mucosa, and in HNSCC. Dissociation curve analysis and sequencing did not validate a specific product for SNAI1 in the used tissue samples.
The CT-values of ZEB1 ranged from 31 to 34 in normal mucosa, and from 30 to 35 in HNSCC. The CTvalues of TWIST ranged from 35 to 37 in normal mucosa, and from 32 to 37 in HNSCC. ZEB1 showed low expression, but its sequence was validated. All PCR products were also amplified and subjected to agarose gel electrophoresis. The electrophoresis image of all PCR products displayed single bands, which were the same size as the one published in the original publications of the primers (Supplementary Table 1).
The % of positive cells and staining intensity of Slug is higher in cases with increased SNAI2 gene expression.
Although, a significant correlation between immunohistochemical Slug staining intensity or the frequency of the stained cells among tumor cells and SNAI gene expression was not possible to state, both the % of Slug positive cells in cancer cell nests as well as the Slug staining intensity were higher in HNSCC cases with upregulated SNAI2 gene expression than in the cases with SNAI2 gene expression level at the normal control mucosa.