Fig 2: KLK6 enzyme contributes to cell invasion in HCT116-shKLK6 model through induction of the EMT.(A). KLK6 enzymatic activity is required for invasive phenotype of HCT116 cells. Upper panel: Cell invasion through Matrigel following transient transfection of KLK6 wt or KLK6 S197A plasmids into HCT116 control clone 1 and shKLK6 clone 3. Cells were seeded in Matrigel invasion chambers 24 h after transfection and were analyzed 48 h later. HCT116 Control clone 1 untransfected and co-transfected with pcDNA3.1 vector served as controls. * p = 0.0004 (shKLK6 +pcDNA3.1 vs shKLK6+wt KLK6), ** p = 0.0008 (shKLK6-3/KLK6 wt vs shKLK6/KLK6) S197A, by t-test. This figure is representative of two independent experiments performed in sextuplets with error bar indicating SD. Middle panel: Secreted KLK6 protein levels in conditioned media by ELISA from Matrigel invasion assay (upper panel), *** p = 0.0002 and ** p = 0.0008 (shKLK6+wt KLK6 vs shKLK6+KLK6 KLK6 S197A), by paired t-test, respectively. Lower panel. Analysis of secreted active TGF-ß2 protein in the conditioned media from Matrigel invasion assay. Figures are representative of two independent experiments performed with triplicates. Error bars indicate SD. (B) Levels of ZO1, E cadherin and Snail in HCT-shKLK6 cell model. RIU: relative intensity units, quantification was done using Image J. Bands of the protein of interest were normalized to ß-actin. The proteins sizes are indicated in kilodalton (kDa). Images are representative of three independent experiments.

Fig 3: Bioinformatic analysis of KLK6 and HMGA2 expression in the CRC patients.(A) Boxplot analysis of KLK6 expression in K-RAS–mutant and non-K-RAS mutant samples in colon adenocarcinomas from the TCGA database. (B) Analysis of KLK6 and HMGA2 expression in 23 TCGA CRC samples stratified by high and low expression of KLK6. Heatmap shows the correlation of KLK6 with HMGA2 expression in these samples. P = 0.02, r = 0.47 by Spearman correlation analysis.

Fig 4: Expression of KLK6 correlates with expression of LIN28B and HMGA2 proteins in colon cancer cells.(A) Expression of LIN28B in HCT116-shKLK6 model and Caco-KLK6 cell model by Western blotting. ß-actin was used as a loading control. RIU: relative intensity units, quantification was done using ImageJ. Bands of the protein of interest were normalized to ß-actin. The protein size is indicated in kDa. Figure is representative of two independent experiments. (B) Expression of HMGA2 by qPCR in HCT116-shKLK6 and Caco-KLK6 isogenic clones. P values were calculated by ANOVA.

Fig 5: Analysis of SMAD signaling and ß-catenin expression in Caco-KLK6 isogenic clones (A) The level of TGF-ß2 transcript in Caco-KLK6 isogenic clones by qPCR; * p < 0.02 (Mock vs Caco-KLK6 clones wt clones 5 and 21, KLK6 S197A clones 1 and 5, by ANOVA). (B) The level of active TGF-ß2 ligand in the conditioned media of Caco-2 isogenic clones expressing the active KLK6 protease (KLK6 wt 5) and mutant KLK6 (KLK6 S197A 5). (C) Analysis of intracellular levels of SMAD proteins in Caco-KLK6 stable isogenic clones by Western blotting. (D) Analysis of the ß-catenin expression in Caco-KLK6 isogenic clones. RIU: relative intensity units, quantification was done using Image J. Bands of phosphorylated SMAD2/3 proteins were normalized to the levels of total SMAD2/3 protein; bands of ß-catenin were normalized to ß-actin. The protein sizes are indicated in kDa. Figure is a representative of two independent experiments.