A MULTI-OMICS APPROACH IDENTIFIES SUBSTRATES AND TARGETS OF ADAM9 IN COLORECTAL CANCER AND OTHER DISEASES

Abstract

Members of the metzincin superfamily of metalloproteinases, including the disintegrin and metalloproteinases (ADAMs) and the matrix metalloproteinases (MMPs), have long been implicated in diseases such as arthritis and cancer. Among all the metzincin metalloproteinases, transmembrane ADAM metalloproteinases have been associated with tumor progression and have emerged as promising targets for cancer treatment. Previous research in our laboratory suggests that ADAM9, a transmembrane ADAM metalloproteinase that is highly expressed in various types of solid tumors, promotes colorectal cancer (CRC) progression. ADAM9 has also been implicated in several other diseases, including neovascularization, retinal degeneration, inflammation, and COVID-19 infection. In this study, to better understand the role ADAM9 plays in CRC and other diseases, I identified new substrates and downstream targets of ADAM9 using a multi-omics approach. To understand how ADAM9 regulates CRC at the transcriptome level, I conducted an RNA-seq analysis on HCT116 CRC cells with ADAM9 knockdown (KD). A total of 351 differentially expressed genes (DEGs), including 94 upregulated genes and 257 downregulated genes, were identified between ADAM9-KD and control samples. My enrichment analysis revealed that the PI3K-AKT signaling pathway, as well as its downstream mTOR signaling pathway and FoxO signaling pathway, were all significantly enriched by the DEGs. Through further investigation, we found that KD of ADAM9 in HCT116 cells reduced AKT activity and inhibited the mTOR signaling but not the canonical Wnt signaling downstream of AKT. We also found that the FoxO signaling was activated upon ADAM9 KD. To understand the mechanism of action for ADAM9 at the cellular proteome level, I performed a TMT-based quantitative proteomics analysis on HCT116 cell lysates with ADAM9 KD. A total of 391 differentially expressed proteins (DEPs), including 143 upregulated proteins and 248 downregulated proteins, were identified between ADAM9-KD and control samples. By integrating the transcriptome and cellular proteome data, I identified the cell-surface DEPs that were upregulated at the cellular proteome level while downregulated or unaltered at the transcriptome level upon ADAM9 KD as potential substrates of ADAM9. From the potential substrates identified, ALCAM and IL10RB were validated to be upregulated by ADAM9 KD, and the regulation was found to be ADAM9 protease activity dependent. We also validated that ADAM17 was downregulated upon ADAM9 KD. To further identify potential substrates and downstream targets of ADAM9, I performed a secretomics analysis to compare the secretome proteins in the serum-free conditioned media of the control and ADAM9-KD HCT116 cells. A total of 300 DEPs, including 179 upregulated proteins and 121 downregulated proteins, were identified at the secretome level. The secretomics data were integrated with the transcriptome and cellular proteome data. 9 DEPs were downregulated at the secretome level, upregulated at the proteome level, and downregulated or unaltered at the transcriptome level upon ADAM9 KD. Among the 9 DEPs, 3 (ICAM5, IGSF8, TMEM132A) are cell-surface proteins and they are very likely to be substrates of ADAM9. From the potential substrates of ADAM9 identified, IGSF8 was validated to be upregulated by ADAM9 KD, and the regulation was found to be ADAM9 protease activity independent. In summary, by integrating RNA-seq and two proteomics analyses (cellular proteome and secretome), I was able to identify two downstream signaling pathways as well as several new substrates and other targets of ADAM9. These substrates and targets may provide important mechanistic insight into the versatile roles of ADAM9 in various diseases.