The Hippo signaling pathway plays a key role in tumorigenesis in different cancer types. We investigated the role of the Hippo effector YAP1 in the tumor immune microenvironment (TIME) of urothelial carcinoma of the bladder (UCB) and evaluated the efficacy of immunotherapy in the context of YAP1 signaling. We performed numerous in vitro and in vivo experiments to determine the role of YAP1 using genetic and pharmacological attenuation of YAP1 activity. Briefly, RNA sequencing was carried out with mouse and human cell lines to identify novel YAP1-regulated downstream targets unbiasedly. We then experimentally confirmed that YAP1 regulates the TIME through the IL-6/STAT3 signaling pathway and varied C-X-C motif chemokine regulation. We analyzed several human sample sets to explore the TIME status in the context of YAP1 expression. Our data indicate that YAP1 attenuation decreases M2 macrophages and myeloid-derived suppressor cells in the TIME compared with YAP1-expressing cells. In summary, this study provides insights into YAP1 signaling as a driver for cancer stemness and an inducer of immunosuppressive TIME. Moreover, the therapeutic efficacy of YAP1 attenuation indicates that combined blockade of YAP1 and immune checkpoints may yield clinical value for treating patients with UCB.

In many regions around the world, including the United States, inorganic arsenic (iAs) contaminates groundwater used for drinking, food production, and irrigation. Although the World Health Organization has set a safety limit of 10 μg/L for arsenic in drinking water, an estimated 200 million people worldwide are still exposed to arsenic concentrations above this threshold. Eliciting a broad range of adverse health effects, arsenic is a known carcinogen classified by the International Agency for Research on Cancer (IARC) and causes increased susceptibility to infectious diseases, highlighting its role as an immunotoxicant. The purpose of this study is to elucidate the effects of arsenic on the innate immune system, namely macrophages, using in vitro exposure models. Bone marrow-derived macrophages (BMDMs) were cultured from adult male and female C57/BL6 mice. These naïve macrophages ("M0" BMDMs) were exposed in vitro to a non-cytotoxic dose of iAs (0.1 μM sodium (meta)arsenite) during the 7 day period of macrophage differentiation and stimulated for 24 hrs with LPS and IFNγ (to induce "M1" pro-inflammatory activation) or IL-4 and IL-13 (to induce "M2" anti-inflammatory activation). In a parallel chronic exposure model, RAW 264.7 (RAW) macrophages were cultured in vitro with iAs for 70 days. Culture supernatant analysis for nitric oxide and cytokine secretion revealed sex-dependent differences in immune response between exposure models, as well as between iAs-exposed and nonexposed macrophages, with and without stimulation. Additionally, iAs-exposed macrophages exhibited increased lipid droplet formation and altered lipidomic and metabolomic profiles, as determined by LC/MS. Flow cytometric analysis further revealed changes in macrophage polarization markers in a sex- and stimulation-dependent manner, with M2-related markers being upregulated in iAs-exposed conditions. Finally, to assess the effects of iAs on macrophages in the context of cancer, we demonstrated that iAs-exposed macrophages displayed increased migration toward cancer cell-conditioned media, and promoted cancer cell proliferation. These results suggest that dysregulated macrophage polarization due to iAs exposure could impact susceptibility to diseases. This research contributes to our understanding of the full spectrum of adverse health effects of iAs exposure and may aid in the development of therapeutics for iAs-induced diseases, including cancer.

Supplementary Figure 3 from <i>N-</i>Methyl-d-Aspartate Receptor Type 2B Is Epigenetically Inactivated and Exhibits Tumor-Suppressive Activity in Human Esophageal Cancer

PDF file - 199KB, RNA was extracted from 18 human NSCLCs and 27 pancreatic directly xenografted low passage tumors. Levels of miR200c were measured by quantitative RT-PCR and mRNA levels of Mig6 and EGFR were determined by Affymetrix expression array. Expression levels of A. miR200c, B. Mig6 mRNA or C. EGFR mRNA transcripts were plotted against erlotinib responsiveness.

PDF file - 2120KB, FAK activity is significantly higher in erlotinib-resistant cells from lung and H and N origin. Total cell lysates were extracted from two pairs of lung (H358/H1703 and Calu3/Calu6) and two pairs of H and N (SCC-S/SCC-R and JHU011/JHU028) cancer cell lines and were subjected to immunoblot analysis with antibodies specific for phosphorylated and total FAK. Beta-actin was used a loading control.

Supp Figure 1. Technical validation of Notch Signaling Pathway DNA Methylation PCR Array.

This is a file of "Supplementary Data".

Supplementary Table 5 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Supplementary Figure 1, Table 1 from Detection of Promoter Hypermethylation in Salivary Rinses as a Biomarker for Head and Neck Squamous Cell Carcinoma Surveillance

Supplementary Figure 3 from <i>N-</i>Methyl-d-Aspartate Receptor Type 2B Is Epigenetically Inactivated and Exhibits Tumor-Suppressive Activity in Human Esophageal Cancer

Supplementary Table S4.The correlation between clinicopathological features and methylation in lung cancer.

Supplementary Figures 1-4 from Mitochondrial Cytochrome B Gene Mutation Promotes Tumor Growth in Bladder Cancer

Supplementary Table 2 from Genome-Wide Promoter Analysis Uncovers Portions of the Cancer Methylome

Flow cytometry gating strategy is outlined. After digestion of tumor infiltrated bladders, samples yielded 1-2 million cells per bladder. A) Live T lymphocyte subpopulations were then gated from spleen tissue, and B) bladder T cells of interest were then selected.

Supplementary Figure S1; Supplementary Tables S1-S3.

5 BCG treated rats, 5 untreated rats, and 20 controls rat were sacrificed and bladders were processed in single cell suspensions. CD4 and CD8 T cells were isolated for each sample and RNA was extracted. A gene expression analysis was subsequently performed on sorted CD4 and CD8 cells from each group using an Affymetrix Rat Gene 1.0 ST Array. MVA plot of sorted (A) CD8 and (B) CD4 T cells from MNU rats treated with BCG vs Untreated tumors. Genes selected from the Gene Ontology term "Immune Response" that have <2SD fold change are highlighted in red were overexpressed with BCG and blue where underexpressed with BCG. (C) Q/Q plot depicting relative fold change after BCG (vs untreated tumor) of genes on sorted CD4 vs CD8 cells.

Supplemental information includes Supplemental Figure S1. The characterization of SOX2 as an oncogene in bladder cancer, Supplemental Figure S2. Stem cell-related molecules associated with SOX2 expression, Supplemental Figure S3. CD24+/CD133+ as a potential marker for SOX2-expressing CSCs, Supplemental Figure S4. COX2/PGE2 signaling pathway in regulating SOX2, Supplemental Figure S5. The association of PGE2 expression with apoptosis in YAP1-LV or YAP1-sh cells, Supplemental Figure S6. Correlation among SOX2, YAP1, and COX2 expression in human UCB samples, Supplemental Figure S7. CSC expansion following CDDP treatment suppressed by combination treatment with verteporfin and celecoxib, Supplemental Figure S8. The EGFR-regulated YAP1 expression via PI3K signaling in basal-type UCB, and Supplementary Materials and Methods.

Supplementary Figure 3 from N-Methyl-d-Aspartate Receptor Type 2B Is Epigenetically Inactivated and Exhibits Tumor-Suppressive Activity in Human Esophageal Cancer

Supplementary Table 2 from Pharmacologic Unmasking of Epigenetically Silenced Genes in Breast Cancer

PDF file - 127KB, Levels of Mig6 protein in the panel of 25 human cancer cell lines with known erlotinib resistance was identified by immunoblot analysis followed by densitometric quantification. The expression level of Mig6 was plotted against IC50 of corresponding cell line.