Fig 2: Impact of FOSL1 inhibition on LAC cell proliferation in vitro and in vivo.(a) Percent cell viability of human wild-type (HCC78, H1437, H1568, H1650, H1703 and H2126) and mutant (H23, H358, H441, A549, H1792, H2009 and H2347) KRAS LAC cell lines with three independent FOSL1 shRNAs. Data are relative to number of cells expressing a GFP shRNA. Relative cell number was assessed by an MTS assay for 4 days and repeated three times. (b) Cell cycle analysis using an EdU incorporation assay of mutant (H358 and H2347) and wild-type (H1568 and H1650) KRAS cell lines expressing an inducible shRNA against GFP or FOSL1. shRNA expression was induced by doxycycline (1 µg ml-1) for 3 days and then cells plated for 24 h before analysis. Data are normalized to cells expressing a GFP shRNA. Result is average of three independent experiments. Error bars correspond to s.d. (c) Analysis of active caspase 3/7 in the same cells as in (b). Data are normalized to cells expressing a GFP shRNA. Result is average of three independent experiments. Error bars correspond to s.d. (d) Average tumour volume of xenografts from H358, H2347, H1568 and H1650 cells expressing an inducible GFP or FOSL1 shRNA (n=8–12 per group). Mice were fed 2 mg ml-1 doxycycline plus 5% sucrose in drinking water when cells were implanted. Error bars correspond to s.e.m. (e) Average tumour volume of xenografts from mouse LAC cells (LSZ3) transduced with a control GPF shRNA (circle) and two shRNAs (square and triangle) against Fosl1 (n=8 per group). Error bars correspond to s.e.m. (f) Average tumour volume change of xenografts from H358 cells with inducible GFP and FOSL1 shRNAs (n=12 per group) relative to start of doxycycline treatment. Doxycycline treatment (2 mg ml-1 doxycycline plus 5% sucrose in drinking water) started at day 18 when tumours had an average volume of 80–100 mm3. Error bars correspond to s.e.m. ***P<0.001. (g) Analysis of Ki67 positive cells in representative areas (n=15 per group) of tumours in (f). Error bars correspond to s.e.m. ***P<0.001. (h) Analysis of cleaved caspase 3 positive cells in representative areas (n=10 per group) of tumours in (f). Error bars correspond to s.e.m. P value obtained using a Student's t-test. *P<0.05; **P<0.01. ***P<0.001. (i) Immunohistochemistry to detect FOSL1 expression in representative sections of the same tumours as in (f). Scale bars 50 µm.

Fig 3: Fosl1 is required in a genetically engineered mouse model of advanced LAC.(a) Representative microCT scans of tumours in KrasLSLG12D/+; Trp53flox/flox; Fosl1+/+ (KP) and KrasLSL-G12D/+; Trp53flox/flox; Fosl1flox/flox (KPF) mice treated with AdCre and allowed to develop tumours for 12 weeks. (b) Representative H and E stained sections of KP and KPF lungs. Scale bar is 6 mm. (c) Dot plot of the quantification of tumour area in KP (n=16) and KPF (n=16) groups (n, number of mice per group). Error bars correspond to s.e.m. P value obtained using a Mann–Whitney test. (d) Dot plot showing mean number of tumours per mouse in KP and KPF mice. Error bars correspond to s.e.m. P value obtained using Student's t-test. (e) Bar graph of the average of tumour size in KP and KPF groups. Error bars correspond to s.e.m. P values obtained using Student's t-test. (f) Kaplan–Meier plot of KP (n=20) and KPF (n=16) mice. P values obtained using the log-rank test (Mantel-Cox). *P<0.05; ***P<0.001.

Fig 4: MUC1 and ERK Cooperate to Drive Association of c-Jun and FRA-1Schematic representation of our proposed mechanism in MUC1 expressing pancreatic cancer cells. Phosphorylation of the MUC1 cytoplasmic tail drives downstream activation of ERK, likely involving the association of MUC1 with receptor tyrosine kinases. Increased ERK activity results in phosphorylation of FRA-1 and promotes dimerization with c-Jun. The transcriptional complex is then stabilized and allows for expression of genes involved in migration, invasion, and overall tumor progression.

Fig 5: FOSL1 is overexpressed in pancreatic ductal adenocarcinoma samples(A) Gene expression data from the NCBI GEO dataset GSE16515 was analyzed for expression of FOSL1 (FRA-1) using microarray expression values. Expression values from tumor and normal samples are presented. Statistical comparison of the groups was performed using the Generalized Estimating Equation (GEE) to account for the paired tumor and normal samples. Analysis revealed a significant increase in FOSL1 expression in the tumor group (p < 0.001) as compared to normal. (B) To confirm tumor samples upregulated FOSL1, 16 paired tumor and normal samples were compared. The log2Fold Change in gene expression for tumor/normal was calculated and plotted. 15 of 16 samples show upregulation in the tumor. 8 of 16 showed an upregulation greater then 2 fold (log2Fold Change > 1) (C) Gene expression data for staged pancreatic tumor specimens was mined using the NCBI GEO dataset GSE42952. The expression of FOSL1 was plotted for each specified staging and metastatic site with absent calls ignored. (D) Immunohistochemistry was performed to evaluate the protein expression of FRA-1 in pancreatic cancer. A heatmap representing relative staining for FRA-1 was generated using R. Scoring was based upon intensity of stain observed only within tumor cells. Representative images for the scoring are presented below the heat map.