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Immune checkpoint therapies exhibit impressive efficacy in some patients with melanoma or lung cancer, but the lack of response in most cases presses the question of how general efficacy can be improved. In addressing this question, we generated a preclinical tumor model to study anti-PD-1 resistance by in vivo passaging of Kras-mutated, p53-deficient murine lung cancer cells (p53R172HΔg/+K-rasLA1/+) in a syngeneic host exposed to repetitive dosing with anti-mouse PD-1 antibodies. PD-L1 (CD274) expression did not differ between the resistant and parental tumor cells. However, the expression of important molecules in the antigen presentation pathway, including MHC class I and II, as well as β2-microglobulin, were significantly downregulated in the anti-PD-1–resistant tumors compared with parental tumors. Resistant tumors also contained fewer CD8+ (CD8α) and CD4+ tumor-infiltrating lymphocytes and reduced production of IFNγ. Localized radiotherapy induced IFNβ production, thereby elevating MHC class I expression on both parental and resistant tumor cells and restoring the responsiveness of resistant tumors to anti-PD-1 therapy. Conversely, blockade of type I IFN signaling abolished the effect of radiosensitization in this setting. Collectively, these results identify a mechanism of PD-1 resistance and demonstrate that adjuvant radiotherapy can overcome resistance. These findings have immediate clinical implications for extending the efficacy of anti-PD-1 immune checkpoint therapy in patients. Cancer Res; 77(4); 839–50. ©2016 AACR.

Purpose: The dismal outcome of esophageal cancer patients highlights the need for novel prognostic biomarkers, such as microRNAs (miRNA). Although recent studies have established the role of miRNAs in esophageal carcinoma, a comprehensive multicenter study investigating different histologic types, including squamous cell carcinoma (SCC) and adenocarcinoma with or without Barrett's, is still lacking.Experimental Design: miRNA expression was measured in cancerous and adjacent noncancerous tissue pairs collected from 100 adenocarcinoma and 70 SCC patients enrolled at four clinical centers from the United States, Canada, and Japan. Microarray-based expression was measured in a subset of samples in two cohorts and was validated in all available samples.Results: In adenocarcinoma patients, miR-21, miR-223, miR-192, and miR-194 expression was elevated, whereas miR-203 expression was reduced in cancerous compared with noncancerous tissue. In SCC patients, we found elevated miR-21 and reduced miR-375 expression levels in cancerous compared with noncancerous tissue. When comparing cancerous tissue expression between adenocarcinoma and SCC patients, miR-194 and miR-375 were elevated in adenocarcinoma patients. Significantly, elevated miR-21 expression in noncancerous tissue of SCC patients and reduced levels of miR-375 in cancerous tissue of adenocarcinoma patients with Barrett's were strongly associated with worse prognosis. Associations with prognosis were independent of tumor stage or nodal status, cohort type, and chemoradiation therapy.Conclusions: Our multicenter-based results highlight miRNAs involved in major histologic types of esophageal carcinoma and uncover significant associations with prognosis. Elucidating miRNAs relevant to esophageal carcinogenesis is potentially clinically useful for developing prognostic biomarkers and identifying novel drug targets and therapies. (Clin Cancer Res 2009;15(19):6192–200)

This figure shows that PD-L1 expression is not quantitatively altered on the anti-PD-1 resistant tumor cells, compared to parental tumor cells.

Supplementary Data from MicroRNA Expression in Squamous Cell Carcinoma and Adenocarcinoma of the Esophagus: Associations with Survival

This figure shows that CD11b+Gr-1+ cells from anti-PD-1 resistant tumors also show downregulation of MHC class I molecules

MicroRNAs are strongly implicated in such processes as development, carcinogenesis, cell survival, and apoptosis. It is likely, therefore, that they can also modulate sensitivity and resistance to anticancer drugs in substantial ways. To test this hypothesis, we studied the pharmacologic roles of three microRNAs previously implicated in cancer biology (let-7i, mir-16, and mir-21) and also used in silico methods to test pharmacologic microRNA effects more broadly. In the experimental system, we increased the expression of individual microRNAs by transfecting their precursors (which are active) or suppressed the expression by transfection of antisense oligomers. In three NCI-60 human cancer cell lines, a panel of 60 lines used for anticancer drug discovery, we assessed the growth-inhibitory potencies of 14 structurally diverse compounds with known anticancer activities. Changing the cellular levels of let-7i, mir-16, and mir-21 affected the potencies of a number of the anticancer agents by up to 4-fold. The effect was most prominent with mir-21, with 10 of 28 cell-compound pairs showing significant shifts in growth-inhibitory activity. Varying mir-21 levels changed potencies in opposite directions depending on compound class; indicating that different mechanisms determine toxic and protective effects. In silico comparison of drug potencies with microRNA expression profiles across the entire NCI-60 panel revealed that ∼30 microRNAs, including mir-21, show highly significant correlations with numerous anticancer agents. Ten of those microRNAs have already been implicated in cancer biology. Our results support a substantial role for microRNAs in anticancer drug response, suggesting novel potential approaches to the improvement of chemotherapy. [Mol Cancer Ther 2008;7(1):1–9]

This figure shows that the immune suppressive MDSCs and regulatory T cell percentages in parental and anti-PD-1 resistant tumor models.

Advances in the understanding of cancer cell biology and response to drug treatment have benefited from new molecular technologies and methods for integrating information from multiple sources. The NCI-60, a panel of 60 diverse human cancer cell lines, has been used by the National Cancer Institute to screen >100,000 chemical compounds and natural product extracts for anticancer activity. The NCI-60 has also been profiled for mRNA and protein expression, mutational status, chromosomal aberrations, and DNA copy number, generating an unparalleled public resource for integrated chemogenomic studies. Recently, microRNAs have been shown to target particular sets of mRNAs, thereby preventing translation or accelerating mRNA turnover. To complement the existing NCI-60 data sets, we have measured expression levels of microRNAs in the NCI-60 and incorporated the resulting data into the CellMiner program package for integrative analysis. Cell line groupings based on microRNA expression were generally consistent with tissue type and with cell line clustering based on mRNA expression. However, mRNA expression seemed to be somewhat more informative for discriminating among tissue types than was microRNA expression. In addition, we found that there does not seem to be a significant correlation between microRNA expression patterns and those of known target transcripts. Comparison of microRNA expression patterns and compound potency patterns showed significant correlations, suggesting that microRNAs may play a role in chemoresistance. Combined with gene expression and other biological data using multivariate analysis, microRNA expression profiles may provide a critical link for understanding mechanisms involved in chemosensitivity and chemoresistance. [Mol Cancer Ther 2007;6(5):1483–91]

Supplementary Data from MicroRNAs modulate the chemosensitivity of tumor cells

This figure addresses the reviewer's questions about the effect of blocking type I IFN on tumor cells and tumor-infiltrating lymphocytes.

All the figure legend for supplementary figures

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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 Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary References from MicroRNA Microarray Identifies Let-7i as a Novel Biomarker and Therapeutic Target in Human Epithelial Ovarian Cancer

Supplementary Table 5 from MicroRNA Signatures in Human Ovarian Cancer

Supplementary Table 3B from Tumor Suppressor Functions of ARLTS1 in Lung Cancers

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

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

MicroRNAs (miR) are a class of small (∼21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1−/− HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3′-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC–treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049–58]

Supplementary Table 3 from MicroRNA Signatures in Human Ovarian Cancer

Supplementary Figure 2 from MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

Supplementary Table 2 from MicroRNA Signatures in Human Ovarian Cancer

We investigated the role of microRNAs (miRNAs) in the pathogenesis of human hepatocellular carcinoma (HCC). A genome-wide miRNA microarray was used to identify differentially expressed miRNAs in HCCs arisen on cirrhotic livers. Thirty-five miRNAs were identified. Several of these miRNAs were previously found deregulated in other human cancers, such as members of the let-7 family, mir-221, and mir-145. In addition, the hepato-specific miR-122a was found down-regulated in ∼70% of HCCs and in all HCC-derived cell lines. Microarray data for let-7a, mir-221, and mir-122a were validated by Northern blot and real-time PCR analysis. Understanding the contribution of deregulated miRNAs to cancer requires the identification of gene targets. Here, we show that miR-122a can modulate cyclin G1 expression in HCC-derived cell lines and an inverse correlation between miR-122a and cyclin G1 expression exists in primary liver carcinomas. These results indicate that cyclin G1 is a target of miR-122a and expand our knowledge of the molecular alterations involved in HCC pathogenesis and of the role of miRNAs in human cancer. [Cancer Res 2007;67(13):6092–9]

Supplementary Table 1 from MicroRNA Signatures in Human Ovarian Cancer

Supplementary Figure 1 from MicroRNA Microarray Identifies Let-7i as a Novel Biomarker and Therapeutic Target in Human Epithelial Ovarian Cancer

B-cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia in the world. Deregulation of the TCL1 oncogene is a causal event in the pathogenesis of the aggressive form of this disease as was verified by using animal models. To study the mechanism of Tcl1 regulation in CLL, we carried out microRNA expression profiling of three types of CLL: indolent CLL, aggressive CLL, and aggressive CLL showing 11q deletion. We identified distinct microRNA signatures corresponding to each group of CLL. We further determined that Tcl1 expression is regulated by miR-29 and miR-181, two microRNAs differentially expressed in CLL. Expression levels of miR-29 and miR-181 generally inversely correlated with Tcl1 expression in the CLL samples we examined. Our results suggest that Tcl1 expression in CLL is, at least in part, regulated by miR-29 and miR-181 and that these microRNAs may be candidates for therapeutic agents in CLLs overexpressing Tcl1. (Cancer Res 2006; 66(24): 11590-3)

Supplemental Figures 1-9 and Tables 1 and 2. Supplemental Table 1: Identification of potential FOXP3-targeted microRNAs in breast cancer cells. Supplemental Table 2: Primer sequences used in this study. Supplemental Figure 1: Expression profiles of IRAK1 and TRAF6 in MCF10A cells with miR-146a/b inhibitors. Supplemental Figure 2: Expression of nuclear FOXP3 in FOXP3-Tet-off MCF7 cells. Supplemental Figure 3: The expression profile of NF-κB target genes after FOXP3 induction in FOXP3-Tet-off MCF7 cells. Supplemental Figure 4: Identification of potential FOXP3-binding sites in promoter regions of miR-146a/b in the FOXP3-Tet-off MCF7 cells by whole-genome ChIP-seq. Supplemental Figure 5: Quantitative analysis of FOXP3-binding sites in the potential promoter region of human miR-146b in FOXP3-Tet-off MCF7 cells. Supplemental Figure 6: Specific binding of FOXP3 to the second forkhead-binding motif (GCAAATA) in the miR-146a promoter. Supplemental Figure 7: MiR-146a/b upregulation by FOXP3 transfection in MDA-MB231 cells. Supplemental Figure 8: The mRNA expression levels of IRAK1and TRAF6 in metastatic breast tumor cells after administration of miR-146a/b inhibitors. Supplemental Figure 9: A potential mechanism by which FOXP3 regulates the NF-κB-miR-146a/b feedback loop through IRAK1 and TRAF6 in tumor growth or regulates CXCR4/MMP9 in tumor metastasis.

Supplementary Figure 1 from MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

Supplementary Methods, Tables 1-4, Figure Legend 1 from Tumor Suppressor Functions of ARLTS1 in Lung Cancers

Supplementary Figure Legends 1-2 from MicroRNAs 221 and 222 Bypass Quiescence and Compromise Cell Survival

Supplementary Figure 1 from Tumor Suppressor Functions of ARLTS1 in Lung Cancers

Supplementary Table 5 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplementary Table 2 from MicroRNA Microarray Identifies Let-7i as a Novel Biomarker and Therapeutic Target in Human Epithelial Ovarian Cancer

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The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but the underlying mechanism still remains largely unknown. Here, we identified a FOXP3–microRNA-146 (miR-146)–NF-κB axis in vitro and in vivo in prostate cancer cells. We observed that FOXP3 dramatically induced the expression of miR-146a/b, which contributed to transcriptional inhibition of IRAK1 and TRAF6, in prostate cancer cell lines. Tissue-specific deletion of Foxp3 in mouse prostate caused a significant reduction of miR-146a and upregulation of NF-κB activation. In addition, prostatic intraepithelial neoplasia lesions were observed in miR-146a–mutant mice as well as in Foxp3-mutant mice. Notably, the NF-κB inhibitor bortezomib inhibited cell proliferation and induced apoptosis in prostate epithelial cells, attenuating prostatic intraepithelial neoplasia formation in Foxp3-mutant mice. Our data suggest that the FOXP3–miR-146–NF-κB axis has a functional role during tumor initiation in prostate cancer. Targeting the miR-146–NF-κB axis may provide a new therapeutic approach for prostate cancers with FOXP3 defects. Cancer Res; 75(8); 1714–24. ©2015 AACR.

Supplementary Table 4A from Tumor Suppressor Functions of ARLTS1 in Lung Cancers

Epithelial ovarian cancer (EOC) is the sixth most common cancer in women worldwide and, despite advances in detection and therapies, it still represents the most lethal gynecologic malignancy in the industrialized countries. Unfortunately, still relatively little is known about the molecular events that lead to the development of this highly aggressive disease. The relatively recent discovery of microRNAs (miRNA), a class of small noncoding RNAs targeting multiple mRNAs and triggering translation repression and/or RNA degradation, has revealed the existence of a new level of gene expression regulation. Multiple studies involving various types of human cancers proved that miRNAs have a causal role in tumorigenesis. Here we show that, in comparison to normal ovary, miRNAs are aberrantly expressed in human ovarian cancer. The overall miRNA expression could clearly separate normal versus cancer tissues. The most significantly overexpressed miRNAs were miR-200a, miR-141, miR-200c, and miR-200b, whereas miR-199a, miR-140, miR-145, and miR-125b1 were among the most down-modulated miRNAs. We could also identify miRNAs whose expression was correlated with specific ovarian cancer biopathologic features, such as histotype, lymphovascular and organ invasion, and involvement of ovarian surface. Moreover, the levels of miR-21, miR-203, and miR-205, up-modulated in ovarian carcinomas compared with normal tissues, were significantly increased after 5-aza-2′-deoxycytidine demethylating treatment of OVCAR3 cells, suggesting that the DNA hypomethylation could be the mechanism responsible for their overexpression. Our results indicate that miRNAs might play a role in the pathogenesis of human EOC and identify altered miRNA gene methylation as a possible epigenetic mechanism involved in their aberrant expression. [Cancer Res 2007;67(18):8699–707]

Supplementary Table 4 from MicroRNA Signatures in Human Ovarian Cancer

Supplementary Figure 2 from MicroRNA Signatures in Human Ovarian Cancer

Supplementary Table 2 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

Supplemental Table 1: Primer sequences used in this study. Supplemental Figure 1: The effect of FOXP3, NF-κB p65-siRNA, and NF-κB inhibitor bortezomib on the growth of DU145 cells.

Supplementary Table 3 from Cyclin G1 Is a Target of miR-122a, a MicroRNA Frequently Down-regulated in Human Hepatocellular Carcinoma

FOXP3 functions not only as the master regulator in regulatory T cells, but also as an X-linked tumor suppressor. The tumor-suppressive activity of FOXP3 has been observed in tumor initiation, but its role during tumor progression remains controversial. Moreover, the mechanism of FOXP3-mediated tumor-suppressive activity remains largely unknown. Using chromatin immunoprecipitation (ChIP) sequencing, we identified a series of potential FOXP3-targeted miRNAs in MCF7 cells. Notably, FOXP3 significantly induced the expression of miR-146a/b. In vitro, FOXP3-induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis. Functional analyses in vitro and in vivo revealed that FOXP3-induced miR-146a/b negatively regulates NF-κB activation by inhibiting the expression of IRAK1 and TRAF6. In ChIP assays, FOXP3 directly bound the promoter region of miR-146a but not of miR-146b, and FOXP3 interacted directly with NF-κB p65 to regulate an miR-146–NF-κB negative feedback regulation loop in normal breast epithelial and tumor cells, as demonstrated with luciferase reporter assays. Although FOXP3 significantly inhibited breast tumor growth and migration in vitro and metastasis in vivo, FOXP3-induced miR-146a/b contributed only to the inhibition of breast tumor growth. These data suggest that miR-146a/b contributes to FOXP3-mediated tumor suppression during tumor growth by triggering apoptosis. The identification of a FOXP3–miR-146–NF-κB axis provides an underlying mechanism for disruption of miR-146 family member expression and constitutive NF-κB activation in breast cancer cells. Linking the tumor suppressor function of FOXP3 to NF-κB activation reveals a potential therapeutic approach for cancers with FOXP3 defects. Cancer Res; 75(8); 1703–13. ©2015 AACR.