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Supplementary Table 1. Characteristics of the African-American prostate cancer patients in the Cleveland Clinic Cohort according to overall survival; Supplementary Table 2. IRDS Probesets (n=49) for the Affymetrix U133A 2.0 array

Supplementary Figure 1. Expression of IFNL4 after transfection of human prostate cancer cell lines with the IFNL4-Halo construct; Supplementary Figure 2. Expression of two interferon signatures, IRG and IRDS, in cultured prostate cancer epithelial cells from 14 African-American and 13 European-American men; Supplementary Figure 3. Association of IFNL4 SNP rs12979860-T allele with decreased overall survival among African-American prostate cancer patients in the Cleveland Clinic cohort (n = 197).

Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer–prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers. Cancer Res; 76(5); 1055–65. ©2015 AACR.

Supplementary Table S1: Clinical characteristics of the study population by smoking status. Supplementary Table S2: Immunoglobulin expression in prostate tumors by smoking status of the patients. Supplementary Table S3: Genes differently expressed between current and never smokers in prostate tumors. Supplementary Table S4: Genes differently expressed between current and never/past smokers in 67 prostate tumors.

Supplementary Table S11: Differentially expressed genes comparing prostate tumors from nicotine-treated TRAMP mice with tumors from untreated mice (reference).

Supplementary Table 9: Genes differently expressed between nicotine-treated and untreated 22Rv1 cells.

Supplementary Table S6: Increased expression of genes regulating synaptic signal transduction in the cancerous prostate of nicotine�treated TRAMP mice.

Supplemetary Table 5: Genes differently expressed between current and never and between current and past/never smokers in the combined cohort of 67 prostate tumors

Purpose: Men of African ancestry experience an excessive prostate cancer mortality that could be related to an aggressive tumor biology. We previously described an immune-inflammation signature in prostate tumors of African-American (AA) patients. Here, we further deconstructed this signature and investigated its relationships with tumor biology, survival, and a common germline variant in the IFNλ4 (IFNL4) gene.Experimental Design: We analyzed gene expression in prostate tissue datasets and performed genotype and survival analyses. We also overexpressed IFNL4 in human prostate cancer cells.Results: We found that a distinct interferon (IFN) signature that is analogous to the previously described “IFN-related DNA damage resistance signature” (IRDS) occurs in prostate tumors. Evaluation of two independent patient cohorts revealed that IRDS is detected about twice as often in prostate tumors of AA than European-American men. Furthermore, analysis in TCGA showed an association of increased IRDS in prostate tumors with decreased disease-free survival. To explain these observations, we assessed whether IRDS is associated with an IFNL4 germline variant (rs368234815-ΔG) that controls production of IFNλ4, a type III IFN, and is most common in individuals of African ancestry. We show that the IFNL4 rs368234815-ΔG allele was significantly associated with IRDS in prostate tumors and overall survival of AA patients. Moreover, IFNL4 overexpression induced IRDS in three human prostate cancer cell lines.Conclusions: Our study links a germline variant that controls production of IFNλ4 to the occurrence of a clinically relevant IFN signature in prostate tumors that may predominantly affect men of African ancestry. Clin Cancer Res; 24(21); 5471–81. ©2018 AACR.

Supplementary Table 7: Genes differently expressed between current and never smokers in microdissected prostate tumors.

Supplementary Figure S1: Weight curves for untreated TRAMP mice and TRAMP mice treated with nicotine for 80 days. Supplementary Figure S2: Ig lambda� and Ig kappa�positive B lymphocytes in cancerous prostate tissue by in�situ hybridization. Supplementary Figure S3: qRT�PCR validation of six genes up�regulated among current smokers. Supplementary Figure S4: Analysis of prostate tumors using Bioconductor limma R. Supplementary Figure S5: Gene Set Enrichment Analysis (GSEA) highlighting common features between smoking�related gene signatures in prostate tumors, nicotine�induced gene signatures in human prostate cancer cells, and gene signatures archived in the GSEA database. Supplementary Figure S6: Expression of various nicotinic acetylcholine receptor (AChR) subunits in human prostate cancer cell lines, prostate tumors, and adjacent non�tumor prostate tissues. Supplementary Figure S7: Nicotine�mediated Akt activation in human immortalized prostate epithelial cells and prostate cancer cell lines. Supplementary Figure S8: Effect of nicotine on the mobility of human prostate cancer cell lines. Supplementary Figure S9: Nicotine enhances cell surface integrin expression and extracellular matrix binding (ECM) of 22Rv1 human prostate cancer cells. Supplementary Figure S10: Nicotine enhances proliferation of RWPE�1 in complete K�SFM medium but not in K�SFM medium without EGF and bovine pituitary extract. Supplementary Figure S11: Nicotine did not enhance proliferation of 22Rv1 and PC�3 human prostate cancer cells. Supplementary Figure S12: Lymphotoxin�β plasma levels in prostate cancer patients (Case) and population�based controls (Control) by smoking status.

Supplemetary Table 8: Genes differently expressed between current and never in microdissected tumors using the Bioconductor limma R package.

Supplementary Table 2 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

MicroRNAs are small noncoding RNAs that regulate the expression of protein-coding genes. To evaluate the involvement of microRNAs in prostate cancer, we determined genome-wide expression of microRNAs and mRNAs in 60 primary prostate tumors and 16 nontumor prostate tissues. The mRNA analysis revealed that key components of microRNA processing and several microRNA host genes, e.g., MCM7 and C9orf5, were significantly up-regulated in prostate tumors. Consistent with these findings, tumors expressed the miR-106b-25 cluster, which maps to intron 13 of MCM7, and miR-32, which maps to intron 14 of C9orf5, at significantly higher levels than nontumor prostate. The expression levels of other microRNAs, including a number of miR-106b-25 cluster homologues, were also altered in prostate tumors. Additional differences in microRNA abundance were found between organ-confined tumors and those with extraprostatic disease extension. Lastly, we found evidence that some microRNAs are androgen-regulated and that tumor microRNAs influence transcript abundance of protein-coding target genes in the cancerous prostate. In cell culture, E2F1 and p21/WAF1 were identified as targets of miR-106b, Bim of miR-32, and exportin-6 and protein tyrosine kinase 9 of miR-1. In summary, microRNA expression becomes altered with the development and progression of prostate cancer. Some of these microRNAs regulate the expression of cancer-related genes in prostate cancer cells. [Cancer Res 2008;68(15):6162–70]

Supplementary Table 4 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

The incidence and mortality rates of prostate cancer are significantly higher in African-American men when compared with European-American men. We tested the hypothesis that differences in tumor biology contribute to this survival health disparity. Using microarray technology, we obtained gene expression profiles of primary prostate tumors resected from 33 African-American and 36 European-American patients. These tumors were matched on clinical variables. We also evaluated 18 nontumor prostate tissues from seven African-American and 11 European-American patients. The resulting datasets were analyzed for expression differences on the gene and pathway level comparing African-American with European-American patients. Our analysis revealed a significant number of genes, e.g., 162 transcripts at a false-discovery rate of ≤5% to be differently expressed between African-American and European-American patients. Using a disease association analysis, we identified a common relationship of these transcripts with autoimmunity and inflammation. These findings were corroborated on the pathway level with numerous differently expressed genes clustering in immune response, stress response, cytokine signaling, and chemotaxis pathways. Several known metastasis-promoting genes, including autocrine mobility factor receptor, chemokine (C-X-C motif) receptor 4, and matrix metalloproteinase 9, were more highly expressed in tumors from African-Americans than European-Americans. Furthermore, a two-gene tumor signature that accurately differentiated between African-American and European-American patients was identified. This finding was confirmed in a blinded analysis of a second sample set. In conclusion, the gene expression profiles of prostate tumors indicate prominent differences in tumor immunobiology between African-American and European-American men. The profiles portray the existence of a distinct tumor microenvironment in these two patient groups. [Cancer Res 2008;68(3):927–36]

Legends for Supplementary Figures 1-6 from Genomic Profiling of MicroRNA and Messenger RNA Reveals Deregulated MicroRNA Expression in Prostate Cancer

Supplementary Table 1 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

Supplementary Figure Legends 1-3 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

Supplementary Figures 1-6 from Genomic Profiling of MicroRNA and Messenger RNA Reveals Deregulated MicroRNA Expression in Prostate Cancer

Supplementary Figure 3 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

Supplementary Methods, Figures 1-10, Table 1 from Human Glioma Growth Is Controlled by MicroRNA-10b

Supplementary Figure 2 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

Supplementary Table 1 from miRNA-7 Attenuation in Schwannoma Tumors Stimulates Growth by Upregulating Three Oncogenic Signaling Pathways

Supplementary Table 3 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

Supplementary Table 5 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

Supplementary Figure 1 from Tumor Immunobiological Differences in Prostate Cancer between African-American and European-American Men

Supplementary Tables 1-4 from Genomic Profiling of MicroRNA and Messenger RNA Reveals Deregulated MicroRNA Expression in Prostate Cancer

Supplementary Figures 1-5 from miRNA-7 Attenuation in Schwannoma Tumors Stimulates Growth by Upregulating Three Oncogenic Signaling Pathways

For nearly a decade, the difficulties associated with both the determination and reproducibility of Ras-dependency indexes (RDIs) have limited their application and further delineation of the biology underlying Ras dependency. In this report, we describe the application of a computational single sample gene set enrichment analysis (ssGSEA) method to derive RDIs with gene expression data. The computationally derived RDIs across the Cancer Cell Line Encyclopedia (CCLE) cell lines show excellent agreement with the experimentally derived values and high correlation with a previous in-house siRNA effector node (siREN) study and external studies. Using EMT signature-derived RDIs and data from cell lines representing the extremes in RAS dependency, we identified enriched pathways distinguishing these classes, including the Fas signaling pathway and a putative Ras-independent pathway first identified in NK cells. Importantly, extension of the method to patient samples from The Cancer Genome Atlas (TCGA) showed the same consensus differential expression patterns for these two pathways across multiple tissue types. Last, the computational RDIs displayed a significant association with TCGA cancer patients’ survival outcomes. Together, these lines of evidence confirm that our computationally derived RDIs faithfully represent a measure of Ras dependency in both cancer cell lines and patient samples. The application of such computational RDIs can provide insights into Ras biology and potential clinical applications.

The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 A resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.

Purpose: Men of African ancestry experience an excessive prostate cancer mortality that could be related to an aggressive tumor biology. We previously described an immune-inflammation signature in prostate tumors of African-American (AA) patients. Here, we further deconstructed this signature and investigated its relationships with tumor biology, survival, and a common germline variant in the IFNλ4 (IFNL4) gene. Experimental Design: We analyzed gene expression in prostate tissue datasets and performed genotype and survival analyses. We also overexpressed IFNL4 in human prostate cancer cells. Results: We found that a distinct interferon (IFN) signature that is analogous to the previously described “IFN-related DNA damage resistance signature” (IRDS) occurs in prostate tumors. Evaluation of two independent patient cohorts revealed that IRDS is detected about twice as often in prostate tumors of AA than European-American men. Furthermore, analysis in TCGA showed an association of increased IRDS in prostate tumors with decreased disease-free survival. To explain these observations, we assessed whether IRDS is associated with an IFNL4 germline variant (rs368234815-ΔG) that controls production of IFNλ4, a type III IFN, and is most common in individuals of African ancestry. We show that the IFNL4 rs368234815-ΔG allele was significantly associated with IRDS in prostate tumors and overall survival of AA patients. Moreover, IFNL4 overexpression induced IRDS in three human prostate cancer cell lines. Conclusions: Our study links a germline variant that controls production of IFNλ4 to the occurrence of a clinically relevant IFN signature in prostate tumors that may predominantly affect men of African ancestry. Clin Cancer Res; 24(21); 5471–81. ©2018 AACR.