Spontaneous metastasis xenograft models link CD44 isoform 4 to angiogenesis, hypoxia, EMT and mitochondria-related pathways in colorectal cancer

Objective: To determine the functional relevance of CD44 isoforms for distant metastasis formation in human colorectal cancer (CRC). Design: We used a pan-CD44 knockdown (kd) approach in a clinically relevant cell line/ xenograft model (HT-29) that spontaneously metastasizes to multiple sites in vivo. Transcriptomics, proteomics and kinomics were used in addition to corresponding validation steps to explain the observed effects. Findings were further corroborated by 3D in vitro culture, tissue microarray and bioinformatics analyses. Results: HT-29 cells mainly express the clinically relevant CD44 isoforms 3 and 4. CD44 kd impairs primary tumor formation and abrogates distant metastasis in vivo. Both CD44 isoforms are induced in the paranecrotic, hypoxic regions of the xenograft primary tumors but are largely absent in the corresponding lung metastases. Tumor angiogenesis is improved in the paranecrosis upon CD44 kd, accompanied by reduced HIF-1α, EMT, and CEACAM5 expression. Vice versa, mitochondrial genes and proteins are induced upon CD44 kd as is oxidative phosphorylation. Hypoxia increases VEGF release from 3D HT-29 spheres, which is strikingly enhanced in CD44 kd spheres. The metastasis-promoting role of CD44 is reflected by an unfavorable prognostic effect of CD44 isoform 4 in patients (as opposed to isoform 3). Accordingly, the genes regulated by the CD44 kd in vivo concordantly overlap specifically with the genes regulated by CD44 isoform 4 in patients. Conclusion: In CRC, CD44 kd (most probably isoform 4) evidently impairs metastasis formation by improving VEGF release and thus angiogenesis in hypoxic conditions, thereby decreasing hypoxia, EMT, stemness, and promoting mitochondrial metabolism. Two types of cells were injected into SCID mice: HT-29 cell lines with shRNA-mediated knockdown of CD44 (shCD44) and control HT-29 cell lines transduced with shRNA against firefly luciferase (shLUC). mRNA was isolated from xenograft tumor tissues (seven samples per group) and analyzed using RNA-seq.