Cadherin 6 Is a New RUNX2 Target in TGF-β Signalling Pathway

<div><p>Modifications in adhesion molecules profile may change the way tumor cells interact with the surrounding microenvironment. The Cadherin family is a large group of transmembrane proteins that dictate the specificity of the cellular interactions. The Cadherin switch that takes place during epithelial-mesenchymal transition (EMT) contributes to loosening the rigid organization of epithelial tissues and to enhancing motility and invasiveness of tumor cells. Recently, we found Cadherin-6 (CDH6, also known as K-CAD) highly expressed in thyroid tumor cells that display mesenchymal features and aggressive phenotype, following the overexpression of the transcriptional regulator Id1. In this work, we explored the possibility that CDH6 is part of the EMT program in thyroid tumors. We demonstrate that CDH6 is a new transforming growth factor-β (TGF-β) target and that its expression is modulated similarly to other EMT mesenchymal markers, both in vitro and in thyroid tumor patients. We show for the first time that CDH6 is expressed in human thyroid carcinomas and that its expression is enhanced at the invasive front of the tumor. Finally, we show that CDH6 is under the control of the transcription factor RUNX2, which we previously described as a crucial mediator of the Id1 pro-invasive function in thyroid tumor cells. Overall, these observations provide novel information on the mechanism of the EMT program in tumor progression and indicate CDH6 as a potential regulator of invasiveness in thyroid tumors.</p> </div

TGF-β signaling in EMT program in thyroid cells.

<p>Schematic representation of the TGF-β dependent EMT program in thyroid cells. Malignant transformation of thyroid cells is accompanied by a constitutive activation of the TGF-β signaling pathway. TGF-β cascade controls the expression of a number of transcription factors (EMT-TF) including RUNX2 that alter the gene expression profile of the cells to activate the EMT program.</p

Analysis of the expression levels of CDH6 splicing variants in thyroid-derived cells.

<p>A) Representative amplification curves of CDH6-L (long isoform; black line) and CDH6-S (short isoform, gray line) in non-treated Nthy.ori 3.1 (left panel), B-CPAP (middle panel) and TPC1 (right panel) obtained by qRT-PCR amplification. Numbers represent the average Ct value +/- s.e.m. of a triplicate amplification. B) qRT-PCR analysis of CDH6-L (left) and CDH6-S (right) in non-treated thyroid-derived cell lines. The bars represent the average fold change of the indicated genes in B-CPAP and TPC1 as compared to Nthy.ori 3.1. Target genes were normalized to the geometric mean of levels of three reference genes: GAPDH, CYPA, GUSB. C-E) qRT-PCR analysis of CDH6-L and CDH6-S in non-treated (NT; black bars) or TGF-β treated (white bars) Nthy.ori 3.1 (C), B-CPAP (D), and TPC1 cells (E). The bars represent the average fold change of the CDH6 variants in TGF-β treated cells as compared to non-treated cells, normalized to the geometric mean of GAPDH, CYPA, GUSB. p-value was calculated by two-tailed Student’s t-test. *** p≤ 0.001; ** p≤ 0.01; * p≤ 0.05.</p

Tumor-derived cell lines display a constitutive EMT-like phenotype.

<p>qRT-PCR analysis of EMT markers (A) (E-CAD; N-CAD; CDH16; TNC; VIM; FN 1) and CDH6 (B) in non-treated thyroid-derived cell lines. The bars represent the average fold change of the indicated genes in tumor cells (B-CPAP, TPC1, WRO, and FTC-133) as compared to thyrocytes (Nthy.ori 3.1), normalized to the geometric mean of levels of three reference genes: GAPDH, CYPA, GUSB. C) Western Blot analysis of E-CAD, N-CAD, FN1 and Actin in non-treated Nthy.ori 3.1; B-CPAP and TPC1 cells. D) Western Blot analysis of phosphorylated ERK, phosphorylated AKT, and Actin in Nthy.ori 3.1 (Left panels); B-CPAP (middle panels) and TPC1 (right panels) cells untreated (NT) or after TGF-β exposure for the indicated times. E) Immunofluorescence staining of SMAD2/3 proteins (green) in Nthy.ori 3.1 (upper panels); B-CPAP (middle panels) and TPC1 (lower panels), non-treated (NT) or after TGF-β exposure for the indicated times. DAPI (Blue) stains the nuclei. Magnification 200X. F-H) qRT-PCR analysis of transcription factors known to partake in the EMT program (SNAI1, SNAI2, ZEB1, TWIST, Id1, and RUNX2) in Nthy.ori 3.1 (E), B-CPAP (F) and TPC1 (G) cells, non-treated (NT) or treated with TGF-β for the indicated times. The bars represent the fold change of the indicated genes in TGF-β treated cells as compared to the non-treated cell levels, normalized to the geometric mean of GAPDH, CYPA, GUSB levels. p-value was calculated by two-tailed Student’s t-test. *** p≤ 0.001; ** p≤ 0.01; * p≤ 0.05. Error bars represent s.e.m. (n=3).</p

Primary and metastatic thyroid tumor cells display a higher expression of EMT-markers than normal thyroid tissue.

<p>A) Scatter plot representation of the relative fold expression of EMT markers (E-CAD, N-CAD, TNC, FN 1, and CDH16) obtained by qRT-PCR in primary tumors (n=15; black circles) and LNMs (n=7; black and white circles) as compared to the respective normal tissue (baseline) from human PTC patients. We were not able to obtain detectable levels of TNC for 5 PTC samples and 2 LNMs. B) Scatter plot representation of the relative fold expression of CDH6 obtained by qRT-PCR in primary tumor (n=15; black circles) and LNMs (n=7; black and white circles) as compared to the respective normal tissue (baseline) from human PTC patients. Target gene expression (A and B) was normalized to the geometric mean of GAPDH, CYPA, GUSB levels. p-value was calculated by two-tailed Student’s t-test. *** p≤ 0.001; ** p≤ 0.01; * p≤ 0.05. C) Representative immunohistochemistry analysis of CDH6 expression (brown) in two PTC samples. A total of 15 PTC samples were analyzed. Invasive front is defined as the boundary between tumor lesion and non-neoplastic thyroid tissue. Hematoxylin (blue). Magnification 100X. The insets show higher magnification of the same field and represent nest of cells infiltrating the tumor capsule. IHC staining was analyzed by light microscopy by two authors (AC, SP). Magnification 200X.</p

TGF-β dependent CDH6 induction is mediated by RUNX2.

<p>A) qRT-PCR analysis of CDH6 levels in non-treated (NT) and TGF-β treated (TGF) Nthy.ori cells, in presence of DMSO (mock, white bars), Actinomycin D (grey bars), cycloheximide (black bars). The bars represent the fold expression of the CDH6 mRNA in the indicated samples normalized to the GAPDH levels. The results are the average of two different replicates. B) qRT-PCR analysis of CDH6 levels in Nthy.ori 3.1 cells non-treated (black bars) or treated with TGF-β (white bars) after transfection with RUNX2 siRNA (right) or control siRNA (left). For both control siRNA and RUNX2 siRNA-treated samples, the bars represent the relative fold change of CDH6 upon TGF-β treatment as compared to non-treated cells. The graphs show one representative experiment. The experiment was replicated three times, obtaining comparable results. C and D) qRT-PCR analysis of RUNX2 and CDH6 mRNA levels in B-CPAP (C) and TPC1 (D) cells non-treated (NT, black bars) or treated with 10mM SB-431542 for 24h (grey bars) or 48h (white bars). Expression levels of CDH6 and RUNX2 were normalized to the geometric mean of GAPDH and CYPA. p-value was calculated by two-tailed Student’s t-test. *** p≤ 0.001; ** p≤ 0.01; * p≤ 0.05.</p

Nthy.ori 3.1 thyrocytes are more responsive to the TGF-β-mediated EMT program than tumor-derived cells.

<p>A-E) qRT-PCR analysis of EMT markers (E-CAD; N-CAD; CDH16; TNC; VIM; FN 1) and CDH6 in non-treated (NT; black bars) or TGF-β treated (5 ng/ml grey bars; 100 ng/ml white bars) thyroid-derived cell lines. The bars represent the average fold change of indicated genes in TGF-β treated cells as compared to non-treated cells, normalized to the geometric mean of levels of three reference genes: GAPDH, CYPA, GUSB. F) Western Blot analysis of E-CAD, N-CAD, FN 1, and Actin in Nthy.ori 3.1 cells, B-CPAP and TPC1 cells non-treated (NT) or treated with 100 ng/ml of TGF-β for 6h and 24h. G, H) qRT-PCR analysis of TGFR1 (G) and TGFR2 (H) in non-treated thyroid-derived cell lines. The bars represent the average fold change of TGFR1 and TGFR2 in tumor cells (B-CPAP, TPC1, WRO, and FTC-133) as compared to thyrocytes (Nthy.ori 3.1), normalized to the to the geometric mean of GAPDH, CYPA, GUSB levels. Error bars represent s.e.m. (n=3). p-value was calculated by two-tailed Student’s t-test. *** p≤ 0.001; ** p≤ 0.01; * p≤ 0.05. Each experiment has been replicated a minimum of two times with comparable results.</p

Additional file 1 of Spatially resolved, high-dimensional transcriptomics sorts out the evolution of biphasic malignant pleural mesothelioma: new paradigms for immunotherapy

Supplementary Material

Tollip interactions.

<p>A) GST-Tollip interaction with ³⁵S-methionine labelled proteins isolated by the yeast two hybrid technique. Lane 1 contains 1/10 input of the ³⁵S-methionine protein used for each interaction; lanes 2 and 3 contain the elution product from incubation of the ³⁵S-proteins with the GST-Tollip and GST-protein alone, respectively.³⁵S-methionine labelled proteins were visualized by autoradiography. Western blot analysis of 293T cells transfected with Ubc9 and HA-Tollip (B), Flag-ARIP3 and HA-Tollip (D), HA-Cystatin B and Ubc9 (C), HA-Cystatin B and Flag-ARIP3 (E). HA-Cystatin B is a ubiquitous protein with antiprotease function, unrelated to Tollip, nor to the inflammatory pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004404#pone.0004404-DiGiaimo1" target="_blank">[28]</a>. Lane 1 contains the protein extract; lane 2 contains the proteins immunoprecipitated with anti-HA abs. In this and in the following figures, “pe.” refers to the protein extract and “ip.” to the immunoprecipitated protein. Staining carried out as indicated under the figures.</p

Tollip and ARIP3 interacting domains.

<p>A) Schematic representation of HA-Tollip deletion mutants. The black box represents the C2 domain (aa 54–179), the grey box represents the CUE domain (aa 229–274). HA-Tollip wt: aa 1–274; Δ1: aa 1–229; Δ2; aa 1–179: Δ3: aa 54–274; Δ4: aa 94–274; Δ5: aa 134–274; Δ6: aa 179–274; Δ7: aa 54–179. In all western blots described in this figure, Lane 1 contains the protein extract and lane 2 the immunoprecipitated proteins. B) Western blot of protein extracts from 293T cells, transfected with Flag-ARIP3 cDNA and with the indicated HA-Tollip deletion mutants, before (lane 1) and after (lane 2) immunoprecipitation with anti-HA abs. The staining is with anti-Flag abs. C) Western blot from the same protein extracts as in B stained with anti-HA abs. D) Schematic representation of the Flag-ARIP3 deletion mutants. The circle represents the SAP domain (aa 11–45), the white box with black stripes represents the RING domain (aa 347–388) and the black box with white stripes represents the AR-ID domain (aa 443–548). Flag-ARIP3 wt: aa 1–572; Δa: aa 1–467; Δb: aa 1–347; Δc: aa 1–169. E) Western blot of protein extracts from 293T cells transfected with HA-Tollip and the indicated Flag-ARIP3 deletion mutants. The protein extract was immunoprecipitated with anti-HA abs and stained with anti-Flag antibodies. F) Western blot of protein extracts from 293T cells transfected with the Flag-PIAS-1 cDNA together with HA-Tollip wt and mutants as indicated. Immunoprecipitation with anti-HA abs; staining with anti-Flag abs. G) Western blot of protein extracts from 293T cells transfected with the Flag-PIASxβ cDNA together with the HA-Tollip wt and mutants as indicated. The protein extract was immunoprecipitated with anti-HA abs and stained with anti-Flag abs. H, I) Western blots of the same samples as in F and G respectively, stained with anti–HA abs.</p