IDDF2021-ABS-0173 Cancer-associated fibroblasts promote colorectal cancer progression through IGF2-IGF1R-YAP1 axis

Background Colorectal cancer (CRC) is one of the common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components. The study aims to elucidate the driving role of CAFs in CRC. Methods Primary CAFs were isolated for RNA-seq analysis, and conditioned medium (CM) was collected from normal fibroblasts (NFs) and CAFs. CRC cells HCT116 and DLD1 were treated with NF-CM or CAF-CM respectively for functional tests. RNA-seq analysis of IGF2-stimulating HCT116 cells was performed to reveal the prominent downstream pathways. qRT-PCR, Western blot, immunofluorescence, and immunohistochemistry were used to validate the gene expression. In vivo studies were performed by subcutaneous xenograft formation model to appraisal the efficacies of small molecule co-administration. Results CAFs-derived CM promoted proliferation, migration, and invasion of HCT116 and DLD1 cells (IDDF2021-ABS-0173 Figure 1A, IDDF2021-ABS-0173 Figure 1B, IDDF2021-ABS-0173 Figure 1C, IDDF2021-ABS-0173 Figure 1D, IDDF2021-ABS-0173 Figure 1E, IDDF2021-ABS-0173 Figure 1F, IDDF2021-ABS-0173 Figure 1G, IDDF2021-ABS-0173 Figure 1H, IDDF2021-ABS-0173 Figure 1I, IDDF2021-ABS-0173 Figure 1J). IGF2 was listed in the top high-expression genes in CAFs compared with NFs, and its predominant receptor IGF1R serves as a prognostic marker to predict the unfordable outcomes of CRC patients (IDDF2021-ABS-0173 Figure 2A, IDDF2021-ABS-0173 Figure 2B, IDDF2021-ABS-0173 Figure 2C, IDDF2021-ABS-0173 Figure 2D, IDDF2021-ABS-0173 Figure 2E, IDDF2021-ABS-0173 Figure 2F, IDDF2021-ABS-0173 Figure 2G, IDDF2021-ABS-0173 Figure 2H, IDDF2021-ABS-0173 Figure 2I, IDDF2021-ABS-0173 Figure 2J, IDDF2021-ABS-0173 Figure 2K). Apart from Akt-mTOR pathway, RNA-seq analysis revealed that YAP1 signaling serves as a prominent downstream modulator to mediate oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq), IGF2 was found to be predominantly secreted by CAFs, while IGF1R-YAP1 was averagely distributed in CAFs and cancer cells. When stimulating IGF1R by rhIGF2, F-actin remodeling, nuclear accumulation of YAP1, and overexpression of YAP1 targets were observed, but these effects were attenuated by either IGF1R depletion or Picropodophyllin (IGF1R inhibitor) administration (IDDF2021-ABS-0173 Figure 3A, IDDF2021-ABS-0173 Figure 3B, IDDF2021-ABS-0173 Figure 3C, IDDF2021-ABS-0173 Figure 3D, IDDF2021-ABS-0173 Figure 3E, IDDF2021-ABS-0173 Figure 3F, IDDF2021-ABS-0173 Figure 3G, IDDF2021-ABS-0173 Figure 3H, IDDF2021-ABS-0173 Figure 3I, IDDF2021-ABS-0173 Figure 3J, IDDF2021-ABS-0173 Figure 3K, IDDF2021-ABS-0173 Figure 3L, IDDF2021-ABS-0173 Figure 3M, IDDF2021-ABS-0173 Figure 3N, IDDF2021-ABS-0173 Figure 3O). By in vivo studies, we found that co-targeting IGF1R and YAP1 by Picropodophyllin and Verteporfin (YAP1 inhibitor) achieved the anti-tumor effects in subcutaneous xenograft and orthotopic implantation models. The synergistic effects were also validated in a patient-derived organoid with IGF1R-YAP1 co-upregulation (IDDF2021-ABS-0173 Figure 4A, IDDF2021-ABS-0173 Figure 4B, IDDF2021-ABS-0173 Figure 4C, IDDF2021-ABS-0173 Figure 4D, IDDF2021-ABS-0173 Figure 4E, IDDF2021-ABS-0173 Figure 4F, IDDF2021-ABS-0173 Figure 4G, IDDF2021-ABS-0173 Figure 4H, IDDF2021-ABS-0173 Figure 4I, IDDF2021-ABS-0173 Figure 4J). Conclusions This study revealed the promoting role of CAFs in CRC progression. The findings highlight the translational potential of IGF2-IGF1R-YAP1 axis, which may serve as a prognostic biomarker and therapeutic target for CRC.