Transcriptome profiling of Native Hawaiian and Japanese CRC-S patients reveals ethnicity-based genetic variability in CRC-S response and progression
Native Hawaiian CRC patients diagnosed with sepsis (CRC-S) demonstrated poor survival outcomes after diagnosis that were considerably lower than Japanese CRC-S patients, with a median survival of 5 and 117 months, respectively (Table 1), suggesting a role for ethnicity-based genetic variability in sepsis mortality risk. We therefore investigated interpatient heterogeneity in the transcriptomic response to sepsis in patients from Native Hawaiian and Japanese ethnic backgrounds (Fig. 1a). Patient and tumor characteristics of study participants are summarized in Table 2. We performed analysis with baseline and tumor characteristics by ethnicity and found that there is no significant correlation of ethnicity with other phenotypes as supported by the frequency counts shown in Table 2. Principal component analysis of global gene expression for CRC tumors revealed two distinct clusters that correlated only with ethnicity, and not with any other factors including sex, age or disease stage (Supplemental Figure S1A–D). Unsupervised hierarchical cluster analysis of global gene expression for CRC tumors revealed distinct expression patterns for Native Hawaiian samples compared to Japanese samples (Supplemental Figure S2). We identified 5472 significant differentially expressed genes between cohorts (FC > 1.5, < − 1.5; FDR < 0.05) with 1058 (19.3%) upregulated and 4414 (80.7%) downregulated in Native Hawaiian CRC-S patients compared to Japanese CRC-S patients (Fig. 1b,c; Supplemental Figure S3). In agreement with Davenport et al.18, we saw no gene expression differences in inflammatory cytokines interleukin 1 beta (IL1B), interleukin 6 (IL6) and 10 (IL10), tumor necrosis factor (TNF), transforming growth factor beta (TGFB), or interferon alpha (IFNA), beta (IFNB) and gamma (IFNG) genes across septic patients. However, pathway analysis of differentially expressed genes identified functional differences related to viral infection, B cell and T cell signaling, systemic inflammatory response/sepsis and septic shock (Fig. 1d,e). Key genes controlling viral infection, systemic inflammatory responses, sepsis, and septic shock were differentially expressed in the Native Hawaiian cohort including RUNX1 (FC = − 2.11, p = 0.001), BCL2L11 (FC = − 2.27, p = 0.002), FOXO3 (FC = − 3.27, p = 1.00E−04) and MAPKAPK2 (FC = − 3.2, p = 2.00E−04).
Genes involved in viral infection, sepsis and septic shock displayed high levels of heterogeneity with significant differential expression in the Native Hawaiian cohort compared to the Japanese group (Fig. 1E). Top disease phenotypes with significantly altered gene expression in Native Hawaiians CRC-S patients include cancer (p = 2.97E−16–2.70E−04), organismal injury and abnormalities (p = 2.97E−16–1.52E−02), protein synthesis (p = 3.18E−09–1.11E−02), infectious disease (p = 3.41E−09–1.32E−02) and cell death and survival (p = 2.31E−07–1.52E−02; Fig. 2a) with significant overall downregulation in genes that affect these phenotypes. Analysis of canonical pathways revealed significant gene alterations in mechanisms of viral exit from host cells (p = 4.52E−04), coronavirus replication (p = 0.005), translation initiation factor EIF2 (p = 7.09E−07) that promotes translation of viral proteins in host cells, and epithelial junction remodeling (p = 4.01E−05; Fig. 2b). The most significant predicted upstream regulators include inflammation response genes (LAPR1; FC = − 3.25, p = 1.74E−09), RPTOR independent companion of MTOR complex 2 (RICTOR; p = 1.51E−07; Fig. 2c) and YAP1 (p = 1.01E−06; Fig. 2d). The transcription regulator MYCN (p = 6.08E−08; Supplemental Figure S4) was also differentially expressed and is primarily involved in cancer specific signaling.
Early response sepsis (ERS) genes involved in viral infection and inflammation are differentially expressed in Native Hawaiian compared to Japanese CRC-S patients
Further pathway analysis of differentially expressed genes identified distinct early response sepsis gene signatures that regulate infection and inflammation and late response sepsis (LRS) genes involved in adherens junction signaling and vascular leak, a component of sepsis progression. ERS signaling pathways including EIF2 (p = 6.59E−07, z-score: − 3.30), integrin-linked kinase (ILK; p = 1.23E−05, z-score: − 1.62), and mTOR (p = 0.042; z-score: 0) were among the most significantly affected canonical pathways in Native Hawaiian CRC-S patients (Fig. 3). A total of 31 genes associated with EIF2 signaling, which was predicted to be inhibited, were identified with ribosomal protein L38 (RPL38; FC = − 16.9, p = 3.09E−05), 5.8S ribosomal N5 (RNA5; FC = − 12.7, p = 0.002), RPL23 (FC = − 11.2, p = 0.004) being the most downregulated and poly(A) binding protein interacting protein 1 (PAIP1; FC = 1.74, p = 6.00E−04) being the only upregulated gene (Fig. 3). Moreover, RPL38 was among the topmost downregulated genes in Native Hawaiian samples compared to those from Japanese patients (Supplemental Figure S5). GSK3B (FC = − 4.38, p = 2.00E−04), MAPK3 (FC = − 1.92, p = 0.002) and ACTA1 (FC = − 2.30, p = 6.00E−04) were downregulated in the Native Hawaiian cohort compared to the Japanese cohort and these genes overlap with ILK, mTOR and VEGF canonical signaling pathways. Among the 32 genes associated with ILK signaling, GSK3B, MAPK3, and VEGFB (FC = − 2.94, p = 1.92E−05) were among the most downregulated genes, while RND2 (FC = 2.16, p = 1.99E−05), PPP2R2B (FC = 1.96, p = 3.03E−05), and dedicator of cytokinesis 1 (DOCK1; FC = 1.89, p = 8.77E−05) were among the most upregulated genes. Moreover, ACTA1, ACTA2 (FC = − 2.77, p = 3.50E−03) and VEGFB cross talk with numerous pathways and signal on the VEGF pathway, which also includes MAPK3, vinculin (VCL; FC = − 2.01, p = 6.00E−04), and SHC1 (FC = − 1.67, p = 0.004). Collectively, these findings support ethnicity-based gene regulation changes in response to sepsis early response genes.
Late response sepsis (LRS) genes involved in permeability regulation are differentially expressed in Native Hawaiian compared to Japanese CRC-S patients
Epithelial junction remodeling is a key mechanism of cancer progression19 yet also essential in the pathophysiological progression from sepsis to septic shock20. These LRS regulated genes control key components of late-stage sepsis including initiation of vascular and intestinal leak. Increased vascular leak further exacerbates MOF by inducing tissue edema20. LRS gene signatures regulating epithelial adherens junction formation and vascular barrier integrity were significantly downregulated in the Native Hawaiian cohort when compared to the Japanese cohort (p = 4.01E−05). LRS genes including VCL, a cytoskeletal remodeling protein involved in junctional remodeling, tubulin beta (TUBB6; FC = − 2.40, p = 0.002), ras-related protein Rab-5B (RAS5B; FC = − 2.08, p = 0.005), tubulin alpha 1b (TUBA1B; FC = − 2.02, p = 0.003), actin alpha 1 (ACTA1; FC = − 2.30, p = 6.00E−04), catenin delta 1 (CTNND1; FC = − 2.27, p = 0.001), tubulin alpha-1C chain (TUBA1C; FC = 2.43, p = 0.002), actin gamma 1 (ACTG1; FC = − 2.09, p = 0.004), protein kinase AMP-activated non-catalytic subunit gamma 2 (PRKAG2; FC = − 3.33, p = 4.00E−04), and vesicle transport through interaction with t-SNAREs 1A (VTI1A; FC = − 2.16, p = 0.001) are all downregulated in Native Hawaiian patient tumors compared to Japanese (Fig. 4a). IPA analysis of candidate biomarkers identified both ERS and LRS genes including GSK3B, WDC2, FOXO3, LDH, MTRNR2L and BCL2L11 (Supplemental Table S1; Fig. 4b). Collectively, such alterations point to a genetic signature whereby genes controlling vascular and intestinal permeability and epithelial junction remodeling in healthy cells are significantly downregulated in Native Hawaiian CRC-S patients compared to Japanese CRC-S patients.