Tree map (hierarchical heat map) depicting affected functional categories based on differentially-expressed transcripts in response to atezolizumab treatment, where the major boxes represent a category of disease and functions (A). CD40 signaling pathways. Using functional assays, BET-IN-1 we confirmed that atezolizumab increases MDA-MB-231 cell apoptosis/necrosis, and reduces their proliferation and viability. Collectively, our findings provide novel insights into the molecular mechanisms/signaling pathways by which atezolizumab exerts inhibitory effects on TNBC, thereby inhibiting EMT/metastasis, tumor growth/survival, and the induction of hypoxia. and 0.05, Figure 2C). gene, metastasis suppressor gene, was upregulated in MDA-MB-231 cells treated with atezolizumab, compared with non-treated cells ( 0.05, Figure 2C). Additionally, genes favoring EMT were significantly downregulated upon atezolizumab treatment, such as ( 0.05, Figure 2C). Genes that inhibit EMT were upregulated BET-IN-1 following atezolizumab treatment, such as and ( 0.05, Figure 2C). Open in a separate window Figure 2 Differentially expressed genes in MDA-MB-231 cells following atezolizumab treatment. Hierarchical clustering of two independent experiments on differentially expressed RNA transcripts from RNA-Seq data. Each column represents a sample and each row represents a transcript. Expression level of each gene in a single sample is depicted according to color scale (A). Heat maps show the fold changes relative to the mean expression of housekeeping genes (B), cell migration/metastasis/adhesion and EMT (C), anti-apoptosis, pro-apoptosis, and cell growth/proliferation (D), DNA repair and hypoxia (E), and signaling transduction (F). Results are from two independent experiments. S1 = sample 1; S2 = sample 2; NT = non-treated cells; T = treated cells with atezolizumab. 2.3. Atezolizumab Downregulates Anti-Apoptotic Genes, Upregulates Pro-Apoptotic Genes, and Downregulates Genes Involved in Cell Growth and Proliferation Our data from RNA-Seq analysis showed that anti-apoptotic genes, and 0.05, Figure 2D), while pro-apoptotic genes, 0.05, Figure 2D). Genes favoring tumor growth and cell proliferation, and were significantly downregulated in treated cells ( 0.05, Figure 2D). On the other hand, tumor suppressor genes and genes inhibiting cell growth, such as 0.05, Figure 2D). 2.4. Atezolizumab Upregulates DNA Repair Genes and Downregulates Genes Related to Hypoxia The involvement of PD-L1 with DNA repair, genomic instability, and hypoxia has not been extensively investigated. Here, we show that genes associated with DNA repair, [29], and [30], were upregulated following atezolizumab treatment ( 0.05. Figure 2E). Additionally, atezolizumab upregulated the gene, which could act as a tumor suppressor gene and a regulator of ATP BET-IN-1 synthesis ( 0.01, Figure 2E). Notably, genes related to hypoxia/ATP synthesis or genes encoding heat shock proteins, including ( 0.05, Figure 2E), and also genes associated with the PI3K, BET-IN-1 MAPK, and NF-kB signaling pathways, BET-IN-1 including 0.05, Figure 2F). In contrast, inhibitors for NF-kB activation, and 0.05, Figure 2F). 2.5. Atezolizumab Downregulates NF-kB, Akt, and CD40 Signaling Pathways Next, we found that about 19% of the genes which were downregulated in atezolizumab-treated cells are associated with EMT, 33% are related to cell migration/invasion and metastasis, 16% are associated with signaling transduction, favoring cell proliferation and EMT, 5% are anti-apoptotic, 8% are related to cell growth and tumor cell proliferation, Rabbit Polyclonal to NSF and 19% are associated with hypoxia (Figure 3A). Selected genes from both upregulated and downregulated panels, including and axes showing Log2 TPM (transcripts per million) of non-treated and treated cells (B). Top significantly affected (?0.5 score ?2.5) pathways based on the upstream regulator analysis (URA). The horizontal bars denote the different pathways based on the score ?2.0) canonical pathways based on ingenuity pathway analysis (IPA). The horizontal bars denote the different pathways based on the score ?2.5, Figure 3C). Using ingenuity pathway analysis (IPA), we showed that atezolizumab regulates other signaling pathways, such as peroxisome proliferator-activated receptor alpha (PPARa)/retinoid X receptor a (RXRa) activation, the sirtuin signaling pathway, the endocannaboid cancer inhibition pathway, the CD40 signaling pathway, the integrin pathway, the relaxin pathway, and the NF-kB and adrenomedullin signaling pathways (1.5 score ?2.0, Figure 3D). Together, these data indicate that atezolizumab in MDA-MB-231 cells is able to downregulate genes and signaling pathways, favoring cell migration/invasion/metastasis, EMT, tumor growth/survival, and genes related to hypoxia. In addition, our data suggest.
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