Fig 2: SRF-dependent tenascin-C, Fgfbp1 and Car12 expression in 4T1 cells. (A) Venn diagram representing a summary of transcript profiling data classifying Mkl1 target genes into three groups: SRF-dependent/SAP-independent, SRF-dependent/SAP-dependent and SRF-independent/SAP-dependent. The number of transcripts, which are more than 2-fold reduced in either 4T1-mutB1 or 4T1-?SAP cells when compared to 4T1-FL cells are shown. (B) Summary listing profiling data, location and functions for the three SRF-dependent/SAP-independent genes, tenascin-C, Fgfbp1 and Car12. Fold reduction and P-value for each of the transcripts in 4T1-mutB1 compared to 4T1-FL cells are shown. References: aBrellier and Chiquet-Ehrismann, 2012; bChiquet-Ehrismann and Chiquet, 2003; cCzubayko et al., 1997; dAbuharbeid et al., 2006; eIvanov et al., 2001; fChiche et al., 2009. (C) qRT-PCR analysis of tenascin-C, Fgfbp1 and Car12 mRNA levels in the stable 4T1 cell lines. Values normalized to Gapdh are expressed relative to the values of 4T1 control cells. Means ± SD from four independent experiments and significant differences to the 4T1 control line, ***P < 0.001 are shown. (D) Immunoblot analysis for tenascin-C, Fgfbp1 and Car12 proteins in the 4T1 cell lines. Secreted tenascin-C protein was detected in cell culture media and BSA from the medium visualized by Ponceau S staining served as loading control. Fgfbp1 and Car12 proteins were detected in whole-cell extracts. Anti-Gapdh served as loading control. Relevant bands and molecular weight markers (in kDa) are indicated on the right and left of each panel, respectively.

Fig 3: Mkl1 induces primary tumor growth and lung metastasis formation. (A) Schematic representation of mouse Mkl1 constructs used for the generation of stable 4T1 cell lines. A full length Mkl1 (FL-Mkl1) construct comprises all functional domains present in the endogenous protein: RPEL1-3, actin binding motifs; B1 and B2, basic domains; Q, glutamine-rich domain; SAP, homology domain with a DNA binding capacity found in SAF-A/B, Acinus, PIAS; LZ, leucine zipper-like domain; TAD, transactivation domain. ?SAP-Mkl1 is a variant lacking the SAP domain, and mutB1-Mkl1 is a full-length protein with a mutated SRF-binding site. All Mkl1 variants contain an RFP-tag at the C-terminus. (B) Immunoblot with mAb65F13 of Mkl1 proteins in whole-cell extracts from 4T1 lines stably transfected with either a control vector encoding RFP alone or with vectors encoding FL-, ?SAP-, or mutB1-Mkl1 proteins. Anti-Gapdh served as loading control. (C) SRF-dependent/SAP-independent proliferation of 4T1 cells in vitro. Proliferation rates of the 4T1 cell lines described in (B) were assessed by BrdU incorporation into newly synthesized DNA immediately after plating (0 h) as well as at 24, 48, 72 and 96 h. Means ± SD from four independent experiments and significant differences to the 4T1 control line at the time point 0, ***P < 0.001, **P < 0.01, *P < 0.05 are shown. (D) SRF-dependent/SAP-independent invasion of 4T1 cells in vitro. Cell invasion through Matrigel-coated filters was assessed and quantified by measuring the area on the underside of the filters covered with cells that have degraded the Matrigel and traversed the filter. Means ± SD from three independent experiments and significant differences to the 4T1 control line, ***P < 0.001 are shown. Representative images of fixed and stained cells of each of the cell lines are shown below the graph. Scale bar, 200 µm. (E) Orthotopic tumor growth of the 4T1 cell lines in mice. Tumor volumes were calculated from caliper measurements taken every 3–4 days. Data represent means ± SD for 5 mice per group. Significant differences to the 4T1 control group, ***P < 0.001, **P < 0.01, *P < 0.05. (F) Metastatic potential of the 4T1 lines presented by a box-and-whisker plot. Number of spontaneous lung metastases in each animal was assessed 20 days after tumor initiation and normalized to the final tumor volume. Data and statistical significance are expressed as in (E). Representative images of H&E-stained lung sections in each of the experimental groups are shown below the plot. Arrows point to metastatic nodules. Scale bar, 500 µm.

Fig 4: Model for the role of SAP-dependent Mkl1 signaling in irradiation (IRR)-induced mammary cancer metastasis. The model summarizes the results from our in vitro and in vivo experiments using the 4T1 breast tumor model. Increased matrix stiffness and dynamic strain trigger SAP-dependent expression of the Mkl1 target genes tenascin-C, Fgfbp1 and Car12 in cultured 4T1 cells. SAP-dependent Mkl1 signaling resulting in the induction of the same genes is also triggered in 4T1 tumors developing within a preirradiated stroma. The increased ECM and in particular collagen deposition in these tumors suggests that they are stiffer and with higher stromal content than tumors grown within nonirradiated mammary tissue. Thereby, we hypothesize that irradiation-induced tissue stiffening mediates SAP-dependent Mkl1 signaling and aggressive progression of mammary tumors locally relapsing after radiotherapy. Our study identifies tenascin-C, Fgfbp1 and Car12 as irradiation-induced Mkl1 target genes with a potential functional implication in the metastatic process. The right part of the model suggests that application of ECM-targeting agents able to decrease the tumor matrix rigidity, such as BAPN (ß-aminopropionitrile) or the hyaluronidase PEGPH20, will impede cancer progression by inhibition of the SAP-dependent Mkl1 signaling.