Fig 2: DDX3 regulated the expression of E-cadherin and ß-catenin through MAPK pathway in CRC cells. (A) Protein expression of DDX3, p-Erk1/2, Erk1/2, E-cadherin, p-ß-catenin (Ser675) and ß-actin in DDX3-NC SW480 cells and DDX3-KD SW480 cells treated with or without 6.25 µmol/L PD98059 by western blot (upper left panel). Protein expression of DDX3, p-MEK1/2, MEK1/2, p-Erk1/2, Erk1/2, p-ß-catenin (Ser675) and ß-actin in DDX3-NC HCT116 cells and DDX3-KD HCT116 cells treated with or without 6.25 µmol/L PD98059 by western blot (lower left panel). Gray analysis of the protein expression in SW480 cells is shown in upper right panel and that in HCT116 cells is shown in lower right panel. ***P<0.001, ns P>0.05 via Student's t-test. (B) Schematic model of DDX3 loss promoting CRC progression by regulating E-cadherin and ß-catenin signaling through the MAPK pathway. Bar graphs are drawn from the mean ± SD of three independent replicates. Abbreviation: CRC, colorectal cancer; KD, knockdown; NC, negative control.

Fig 3: Cancer-associated PPP6C mutations decrease phosphatase activity against MEK1/2(A) Frequencies of PPP6C mutations reported in melanomas. Data are from nonoverlapping melanoma studies in cBioportal (Cerami et al., 2012; Gao et al., 2013).(B) PPP6C residues mutated in cancer are shown in spacefill representation modeled on the X-ray crystal structure of PPP5C in complex with a peptide substrate (PDB: 5HPE). Bound peptide (cyan) and catalytic metal ions (gray spheres) are shown.(C) PPP6C+/+ and PPP6C-/- 501mel cells were transduced to stably express GFP (–), WT PPP6C, or the indicated PPP6C mutants. Cells were lysed and assessed by immunoblot for phosphorylated and total MEK and ERK. Phospho/Total MEK (black) and ERK (blue) signal ratios were quantified and normalized to the GFP-expressing PPP6C+/+ samples. Mean values ± SD are shown, n = 5. Significance is shown in comparison to PPP6C+/+ cells expressing GFP. *p < 0.05, **p < 0.01, ***p < 0.001, paired t test.(D) Cells from (C) were treated with 100 ng/mL nocodazole for 24 h. Mitotic cells were lysed and assessed by immunoblot for phosphorylated and total Aurora A. Phospho-Aurora A/Total Aurora A signal ratios were quantified (n = 5) and significance determined as in (C). *p < 0.05, **p < 0.01, paired t test.(E) Cells from (C) were cultured in media containing DMSO vehicle alone or the indicated trametinib concentration for 2 weeks and stained with crystal violet. Clonogenic growth was analyzed by ColonyArea in ImageJ and normalized to GFP-expressing PPP6C+/+ samples, n = 3.

Fig 4: Correlation of HPIP expression with components of EGFR-RAS-ERK signaling and survival in patients with lung cancer.(A) Correlation of HPIP expression with levels of RAS-GTP and phosphorylated RAF1, MEK1/2, and ERK1/2 as well as expression of RAS and HIF-1a in 40 lung cancer samples. Representative immunoblot with the indicated antibodies is shown. Immunoblots were assembled from different membranes. Statistical significance was assessed by Pearson’s correlation test. (B) The disease-free and overall survival curves related to low and high expression of HPIP were analyzed in 240 patients with lung cancer using the Kaplan-Meier method. (C) A proposed model underlying the role of HPIP in regulating the EGFR-RAS-ERK signaling pathway. HPIP interacts with all classical components of the EGFR-RAS-ERK pathway and forms two complexes with the components as indicated, promoting the expression of downstream target genes (e.g., HIF-1a and c-Myc), aerobic glycolysis, and tumor growth.

Fig 5: HPIP is required for EGFR-RAS-ERK signaling–mediated promotion of cancer cell proliferation and growth in vitro and in vivo.(A) The proliferation curve of HPIP WT or KO A549 or ZR75-1 cells or HPIP KO A549 or ZR75-1 cells stably infected with HPIP or EV and treated with EGF (100 ng/ml) or PBS. (B) The proliferation curve of HPIP WT or KO A549 or ZR75-1 cells stably infected with Flag-tagged KRAS or MEK1 or EV. (C) The proliferation curve of A549 or ZR75-1 cells stably infected with Flag-tagged KRAS or MEK1 and treated with or without TXX-1-10 (10 µM). Data shown are means ± SD of three independent experiments (A) to (C). Statistical significance was assessed by two-tailed Student’s t test. **P < 0.01. (D) The growth curve of xenograft tumors derived from HPIP WT or KO A549 cells stably infected with Flag-tagged KRAS or MEK1 or EV. Tumor volumes were presented as means ± SD (n = 6). Representative immunoblot with the indicated antibodies was shown in the representative excised tumors from the fourth column in each group. **P < 0.01 at day 28. (E) The growth curve of xenograft tumors derived from A549 cells stably infected with Flag-tagged KRAS or MEK1 and treated with TXX-1-10 as indicated. Representative immunoblot with the indicated antibodies was shown in the representative excised tumors. **P < 0.01 at day 28. Immunoblots were assembled from different membranes (D) and (E).