Fig 2: There is a systemic activin response to PDAC. (A, B) Inhba or Inhbb mRNA expression by quantitative RT–PCR in organs harvested at the study endpoint from sham control and orthotopic KPC32043 tumour-bearing mice. Expression values in all organs for each gene were normalized to the value from the control liver. IHC for activin A and activin B in organs from mice with (C) orthotopic KPC32043 tumours, (D) orthotopic KPC32908 tumours, or (E) genetic PDAC (KPC mice). Data are presented as mean ± SEM. Scale bar: 100 µm.

Fig 3: Activin-A promotes anchorage-independent growth but not proliferation of breast cancer cells. (a) Bromodeoxyuridine (BrdU) incorporation assay following activin-A treatment of MCF7 (i) and MDA-MB-231 (iii) cells. Overexpression of INHBA in MCF7 cells (ii) and knockdown of INHBA by small hairpin RNA (shRNA) in MDA-MB-231 cells (iv). MCF-7 cells but not MDA-MB-231 cells show mild inhibition in proliferation on activin-A treatment. (b) Treatment of MCF-7 cells with activin-A results in reduced colony growth (i), but MCF-7 clones overexpressing activin-A show higher colony-forming ability (ii). Treatment of MDA-MB-231 cells with activin-A in soft agar does not show any effect (iii) and stable knockdown of activin-A in MDA-MB-231 cells results in a significant decrease in colony formation (iv).

Fig 4: Inhibin beta A subunit (INHBA) as the major downstream effector in LIF increases oral cancer progression. (A) Heatmap of mRNA expression profile in Cal27/pLKO and Cal27/shLIF stable clones. (B) Gene set enrichment analysis (GSEA) showed the enrichment of metastatic genes in Cal27/pLKO versus Cal27/shLIF cells. (C) Real-time PCR analysis of BUB1, BIRC5, INHBA, CD44, and UBE2C mRNA expression in Cal27/pLKO and Cal27/shLIF cells (** p < 0.01). (D) Reverse transcription PCR analysis of INHBA mRNA expression in CA9-22 and HSC3 cells transiently transfected LIF-expressed or shLIF plasmids. (E) Cal27/pLKO and Cal27/shLIF stable clones were seeded and transiently transfected with 3 µg of control plasmid or various concentrations of INHBA plasmids and incubated for 48 h, then subcultured in a Boyden chamber overnight. Cell motility toward the lower face of the filter was observed and quantified (** p < 0.01). (F) Western blot analysis of INHBA protein expression in CA9-22 cells after they were treated with rLIF. ß-actin was used as an internal control.

Fig 5: Expression of activin and correlation with breast tumor progression. (a) Quantitative PCR analyses of INHBA, SMAD2, FST, and BMP2 expression in breast tumors compared with that in normal breast tissues. It is worth noting the significant increase in the expression of INHBA (activin-A) and Smad2. (b) Immunohistochemistry of activin-A (i), pSMAD2 (ii), pSMAD3 (iii), and BMP2 (iv) in normal and breast tumor sections. Breast tumors show higher levels of activin-A, pSMAD2, and pSMAD3, whereas normal samples have higher levels of BMP2 compared with tumor samples. Each graph below shows the intensity score of individual normal and tumor sample on a scale of 0 to 3. The statistical significance is indicated in the representative graph. (c) GOBO gene set analysis shows that INHBA expression inversely correlates with overall survival (OS) of high-grade breast cancer patients (ii). In addition, INHBA expression correlates inversely with the distant metastasis-free survival (DMSF) of breast cancer patients (iii and iv). (d) GOBO box plot expression analysis shows that expression levels of negative regulators of activin signaling pathway, FST and TGFßR3, decrease progressively from grade 1 to grade 3.