Non-Canonical Activin A Signaling Stimulates Context-Dependent and Cellular-Specific Outcomes in CRC to Promote Tumor Cell Migration and Immune Tolerance

Abstract

We have shown that activin A (activin), a TGF-β superfamily member, has pro-metastatic effects in colorectal cancer (CRC). In lung cancer, activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. Here, we hypothesized that activin exerts cell-specific effects in the tumor microenvironment (TME) of CRC to promote anti-tumoral activity of immune cells and the pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. We generated an Smad4 epithelial cell specific knockout (Smad4-/-) which was crossed with TS4-Cre mice to identify SMAD-specific changes in CRC. We also performed IHC and digital spatial profiling (DSP) of tissue microarrays (TMAs) obtained from 1055 stage II and III CRC patients in the QUASAR 2 clinical trial. We transfected the CRC cells to reduce their activin production and injected them into mice with intermittent tumor measurements to determine how cancer-derived activin alters tumor growth in vivo. In vivo, Smad4-/- mice displayed elevated colonic activin and pAKT expression and increased mortality. IHC analysis of the TMA samples revealed increased activin was required for TGF-β-associated improved outcomes in CRC. DSP analysis identified that activin co-localization in the stroma was coupled with increases in T-cell exhaustion markers, activation markers of antigen presenting cells (APCs), and effectors of the PI3K/AKT pathway. Activin-stimulated PI3K-dependent CRC transwell migration, and the in vivo loss of activin lead to smaller CRC tumors. Taken together, activin is a targetable, highly context-dependent molecule with effects on CRC growth, migration, and TME immune plasticity.

Keywords: activin A; colorectal cancer; digital spatial profiling; metastasis; tumor microenvironment.

Figure 1

Ablation of Smad4 in APC mice leads to decreased survival and is associated with increased activin, pAKT, and CD4. Colon tumors of Smad4−/− mice show significantly increased (A) activin, (B) pAKT, and (C) CD4 compared to Smad4−/+ and Smad4+/+ mice using histopathology. (D) Kaplan–Meier survival curves of Ts4Cre/cApcfl/+/Smad4+/+ (WT) (blue), Ts4Cre/cApcfl/+/Smad4+/− (het) (black), and Ts4Cre/cApcfl/+/Smad4−/− (KO) (magenta) show the loss of survival with ablation of Smad4 (log-rank test p = 0.0018), a downstream signaling molecule of canonical SMAD signaling. Data analyzed via ordinary, one-way ANOVA with Tukey multiple comparisons test (A–C) or Log-rank (Mantel–Cox) test (D) (* p < 0.05, ** p < 0.01; n = 4, 7, and 12).

Figure 2

Pan tumoral activin or TGF-β expression in IHC staining is associated with improved patient outcome in stage II or III CRCs. A total of 1055 patients with stage II or stage III CRC from the QUASAR 2 cohort were stained for activin, TGF-β, and CD4 using IHC. (A) Confirming the validity of the cohort, tumoral CD4 is associated with better survival (blue; p = 0.0069; n = 990). (B) Combined low activin and high TGF-β levels are associated with better outcome in CRC patients (dark blue) as was low TGF-β and high activin (light blue). Low activin and low TGF-β are associated with worse outcome (black; p = 0.0214; activin n = 1042; TGF-β n = 971).

Figure 3

Activin co-localizes with both tumor and immune cells in the TME to generate activin-dependent signaling compartments via DSP. CRC patient tumor samples from the QUASAR 2 clinical trial were stained with four fluorescent markers (PanCK, green; DNA, blue; CD45, yellow; and activin, red) and were separated into stroma or tumor-containing samples using DSP. (A,B) Representative regions of interest (ROI) in the stroma, (C,D) which were separated into two distinct areas of illumination (AOI) prior to collection and quantification: activin positive (pink) and activin negative (gray). (E,F) Representative ROIs in the tumor, (G,H) separated into activin (+)/(−) AOIs. (I) Activin (+) areas of tumor samples in the stroma show the highest heat signature for several markers of T-cell suppression when compared to activin (−) areas. A similar trend was observed in activin (+) areas of the tumor. (J) Activin (+) areas in the tumor and stroma have higher levels of PI3K/AKT signaling proteins when compared to activin (−) areas. (K) Several markers of the stroma, including immune cell markers, are highest in the stroma independent of activin co-localization. (L) The tumor marker PanCK is highest in tumoral compartments, indicating successful classification of tumor vs. stroma. Additionally, several markers of APC activation appeared to be highest in activin (+) areas on the stromal regions. Data are expressed as log2 normalized counts, n = 8, 10, 18, and 20.

Figure 4

Markers of tumoral and stromal compartments confirm successful segregation of regions via DSP with an increase in CD45 observed in activin (−) regions of the stroma. (A) Volcano plot indicating that several proteins, including CD45, α-SMA, and PanCK, are significantly differentially expressed across the tumoral and stromal compartments in the TME. (B) Volcano plot displaying several proteins that are significantly differentially expressed in the stromal compartment of the TME, which is dependent upon activin co-localization. Analysis of the CRC tissue sections across activin (+) and (−) areas within tumoral and stromal compartments identified significant changes in expression of several proteins found in the stroma and tumor of the TME, including (C) ɑ-SMA, (D) PanCK, (E) CD45, (F) CD4, and (G) CD8. Data were analyzed via linear mixed modeling with Benjamini–Hochberg multiple-correction test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 8, 10, 18, and 20).

Figure 5

Activin co-localization is associated with T-cell exhaustion and increased APC activation markers in DSP-analyzed images. Several markers of T-cell exhaustion were significantly upregulated in activin (+) areas in both the tumoral and stromal compartment of the TME, including (A) CTLA-4, (B) FOXP3, and (C) CD25. Significant increases in the T-cell exhaustion marker (D) PD-1 were only observed in activin (+) areas in the tumor. Additionally, increases in markers of APC activation, (E) CD80 and (F) CD40, were upregulated in activin (+) AOIs in both tumoral and stromal compartments of the TME. Data were analyzed via linear mixed modeling with Benjamini–Hochberg multiple-correction testing [34] (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 8, 10, 18, and 20).

Figure 6

Activin-induced PI3K signaling enhances tumoral growth and metastasis. (A–C) Activin co-localization was associated with increased expression of several PI3K/AKT markers, including PLCG1, Phospho-PRAS40, and Phospho-Tuberin in the DSP analysis. (D) In vitro activin stimulated migration of ACVR2A-expressing HCT116+chr2 CRC cells, which was ablated in the presence of the PI3K inhibitor LY294002 but not the MAPK inhibitor U0126. (E) Mice injected with CRC cells which have reduced activin production develop significantly smaller tumors at day 23 post-inoculation. (F) Total tumor growth over time is significantly reduced in mice receiving activin KD CRC cells. DSP data analyzed via linear mixed modeling with Benjamini–Hochberg multiple-correction testing; migration data analyzed via ordinary one-way ANOVA with Tukey multiple comparisons test; tumor growth data analyzed via two-way regular ANOVA with effect of time, cell type, and interaction with Sidak’s multiple comparisons test for post-hoc analysis; AUC data analyzed via ordinary two-tailed student’s t-test (* p < 0.05, ** p < 0.01, **** p < 0.0001; (A–C) n = 8, 10, 18, and 20; (D) n = 18, 19, and 20; (E,F) n = 5).