Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas

Abstract

Interferon Regulatory Factor 6 (IRF6) and Grainyhead Like Transcription Factor 3 (GRHL3) are transcription factors that orchestrate gene regulatory networks required for the balance between keratinocyte differentiation and proliferation. Absence of either protein results in the lack of a normal stratified epidermis with keratinocytes failing to stop proliferating and to terminally differentiate. Numerous pathological variants within IRF6 and GRHL3 have been identified in orofacial cleft-affected individuals and expression of the two transcription factors has been found to be often dysregulated in cancers. However, whether orofacial cleft-associated IRF6 and GRHL3 variants in patients might also affect their cancer risk later in life, is not clear yet. The fact that the role of IRF6 and GRHL3 in cancer remains controversial makes this question even more challenging. Some studies identified IRF6 and GRHL3 as oncogenes, while others could attribute tumor suppressive functions to them. Trying to solve this apparent conundrum, we herein aimed to characterize IRF6 and GRHL3 function in various types of carcinomas. We screened multiple cancer and normal cell lines for their expression, and subsequently proceeded with functional assays in cancer cell lines. Our data uncovered consistent downregulation of IRF6 and GRHL3 in all types of carcinomas analyzed. Reduced levels of IRF6 and GRHL3 were found to be associated with several tumorigenic properties, such as enhanced cell proliferation, epithelial mesenchymal transition, migration and reduced differentiation capacity. Based on our findings, IRF6 and GRHL3 can be considered as tumor suppressor genes in various carcinomas, which makes them potential common etiological factors for cancer and CLP in a fraction of CLP-affected patients.

Keywords: GRHL3; IRF6; cancer; differentiation; orofacial cleft; tumor suppressor.

Figure 1

IRF6 and GRHL3 expression in healthy human adult tissues and cells (A) QPCR analysis of IRF6 and GRHL3 in a panel of healthy human adult tissues. Color code refers to tissue groups introduced in Supplementary Figure 1A . (B) IHC for IRF6 as well as a representative H&E staining of a set of normal human tissues. Note that IRF6 exhibits nuclear as well as cytoplasmic localization. Scale Bar: 50 µm. (C) QPCR analysis of IRF6 and GRHL3 in a panel of healthy human cell strains/lines. not detectable (cT > 32) (D) Immunoblot for the proteins IRF6 and E-Cadherin in a set of human cell strains/lines. Note the typical IRF6 double-band indicative of its phosphorylated and non-phosphorylated form, respectively. Full-length blots are shown in Supplementary Figure 6 kDa, kilo Daltons.

Figure 2

IRF6 and GRHL3 downregulation in cancer cell lines (A) Panels of cancer cell lines and corresponding normal cells (striped boxes) were screened for IRF6, GRHL3, and CDH1. Note that RNA was extracted from all cell cultures at a cell density of approximately 70% confluency kDa, kilo Daltons. * p < 0.05 control vs. cancer cell lines; # not detectable (c _T > 32). HME: human melanocytes. (B) Immunoblots for IRF6 and E-Cadherin in a subset of the analyzed cancer cell lines and controls. Full-length blots are shown in Supplementary Figure 6 . HME: human melanocytes. (C) Brightfield (BF) pictures, IRF6 staining, as well as the merge of IRF6 (green) with actin (red) and cell nuclei (blue) is shown for breast and lung cancer, skin and oral SCC samples and their corresponding normal controls. Note that IRF6 is predominantly expressed in the cytoplasm of the various cells. Scale bar: 25 µm.

Figure 3

IRF6 and GRHL3 overexpression impairs cancer cell proliferation and migration (A) Immunoblots for IRF6 and GRHL3 confirming their overexpression. Full-length blots are shown in Supplementary Figure 6 . kDa, kilo Daltons. (B) Cell growth of empty vector (white)-, IRF6 (black)-, and GRHL3 (gray)- transduced T47D (left) and SCC-68 cell lines (right) shows reduced number of cells by the enhanced presence of IRF6 and GRHL3. * p < 0.05 control vs. IRF6 and GRHL3 (C) qPCR analysis of the proliferation marker KI67 in transduced T47D and SCC-68 cell lines. * p < 0.05 control vs. IRF6 and GRHL3. (D) Transduced cells were analyzed for the expression of EMT markers CDH1, CDH2, VIM, SNAIL1, TWIST2. not detectable (cT > 32). (E) E-Cadherin, N-Cadherin, and Vimentin were analyzed by immunoblots in control and IRF6 and GRHL3 transduced SCC-68 cells. Quantification of the blots is shown below the immunoblots. * p < 0.05 control vs. IRF6 and GRHL3 for N-Cadherin and Vimentin. Full-length blots are shown in Supplementary Figure 6 . kDa, kilo Daltons. (F) Representative live imaging pictures at t=0 h (left) and t=10 h (right) after scratching SCC68 control, SCC68/IRF6, and SCC68/GRHL3. White lanes indicate the migrating fronts on each side of the cell-free gap (left). Quantification of the closure of the cell-free gap is shown on the right. Note a significantly reduced migratory behavior of SCC-68/IRF6 and SCC-68/GRHL3 compared to control starting from t=2 h * p < 0.05 control vs. IRF6 and GRHL3. Scale bar: 500 µm. Movies of the various cell lines are shown in Supplementary Movies 1–3 .

Figure 4

Overexpression of IRF6 and GRHL3 induces differentiation. (A) The differentiation status of three normal cell strains compared to three SCC lines was assessed by qPCR for a panel of differentiation markers (IRF6, GRHL3, IVL, FLG, K10, LOR, and TGM1). The data are shown as a heatmap. Scale: light green – low gene expression; red – high gene expression. (B) IF staining for TGM1, IVL, LOR, and FLG in PA-Ep (non-cancerous) vs. SCC-68 indicates less differentiated cells in the SCC-68 cultures. Scale bar: 50 µm. Note that staining for LOR and TGM1 resulted in a nuclear background staining ( Supplementary Figure 5 ). (C) The same healthy cell strains and SCC cell lines were analyzed in low density (LD) and their corresponding high density (HD) cultures, and the same set of differentiation genes was analyzed by qPCR. The results are shown as heatmap of the fold inductions (HD vs. LD). Scale: light green – low gene induction; red – high gene induction. (D) The effect of forced expression of IRF6 (black) and GRHL3 (gray) compared to control SCC-68 (white) on differentiation markers (K10, FLG, and LOR) was determined by qPCR. * p < 0.05 control vs. IRF6 and GRHL3. (E) Brightfield pictures and corresponding pictures with actin staining indicate the presence of differentiating cell groups (dashed yellow line in the brightfield images and asterisks in the actin staining) in SCC68 cells transduced with IRF6 or GRHL3. Scale bar: 50 µm. (F) K10 staining in dense cultures of transduced SCC-68 cells (left) and its quantification (right). Note the presence of significantly more K10-positive cells in SCC-68/IRF6 and SCC-68/GRHL3 compared to SCC-68 control. Scale bar: 50 µm. * p < 0.05 control vs. IRF6 and GRHL3.