High-mobility group box 1 (HMGB1) is an abundant chromatin-associated nuclear protein and released into the extracellular milieu during liver ischemia-reperfusion (I/R), signaling activation of proinflammatory cascades. Because the intracellular function of HMGB1 during sterile inflammation of I/R is currently unknown, we sought to determine the role of intracellular HMGB1 in hepatocytes after liver I/R. When hepatocytespecific HMGB1 knockout (HMGB1-HC-KO) and control mice were subjected to a nonlethal warm liver I/R, it was found that HMGB1-HC-KO mice had significantly greater hepatocellular injury after I/R, compared to control mice. Additionally, there was significantly greater DNA damage and decreased chromatin accessibility to repair with lack of HMGB1. Furthermore, lack of hepatocyte HMGB1 led to excessive poly(ADP-ribose)polymerase 1 activation, exhausting nicotinamide adenine dinucleotide and adenosine triphosphate stores, exacerbating mitochondrial instability and damage, and, consequently, leading to increased cell death. We found that this was also associated with significantly more oxidative stress (OS) in HMGB1-HC-KO mice, compared to control. Increased nuclear instability led to a resultant increase in the release of histones with subsequently more inflammatory cytokine production and organ damage through activation of Toll-like receptor 9. Conclusion: The lack of HMGB1 within hepatocytes leads to increased susceptibility to cellular death after OS conditions.

Background-Intra-tumor heterogeneity implies that sub-populations of cancer cells that differ in genetic, phenotypic, or behavioral characteristics coexist in a single tumor 1,2. Tumor heterogeneity drives progression, metastasis and treatment resistance, but its relationship with tumor infiltrating immune cells is a matter of debate where some argue that tumors with high heterogeneity may generate neo-antigens that attract immune cells, and the others claim that immune cells provide selection pressure that shapes tumor heterogeneity 3,4. Here we sought to study the association between tumor heterogeneity and immune cells in a real-world cohort utilizing The Cancer Genome Atlas (TCGA). Methods-Mutant Allele Tumor Heterogeneity (MATH) was calculated to estimate intra-tumoral heterogeneity, and immune cell compositions were estimated by CIBERSORT. Survival analyses were demonstrated by Kaplan Meir curves. Results-Tumors with high heterogeneity (High MATH) associated with worse overall survival (p=0.049) as well as ER+ (p=0.011) and non-triple negative tumors (p=0.01). High MATH tumors associated with less infiltration of anti-tumor CD8 (p<0.013) and CD4 T cells (p<0.00024), more tumor promoting regulatory T cells (p<4e-04), lower expression of T cell exhaustion markers; PDL-1 (p=0.0031), IDO2 (p=0.34), ADORA2A (p=0.018), VISTA (p=0.00013), and CCR4 (p<0.00001), lower expression of cytolytic enzymes granzyme-A (p=0.0056) and perforin 1 (p=0.053) as well as low cytolytic activity score (p=0.0028). Conclusions-High heterogeneity tumors are associated with less immune cell infiltration, less activation of the immune response, and worse survival in breast cancer. Our results support the notion that tumor heterogeneity is shaped by selection pressure of tumor infiltrating immune cells.

Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1-TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1's release from hypoxic hepatocytes in vitro and thereby weakened HMGB1's activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury. Figure 5. Eritoran treatment suppresses HMGB1 release in vivo. (A) Nuclear and cytoplasmic HMGB1 levels were determined by Western blot and quantitative densitometry analysis of the protein expressions in eritoran-treated mice and control mice that underwent ischemia and 6 h of reperfusion determined. Hepatic protein lysates from ischemic lobes were obtained; each lane represents a separate animal. The blots shown are representative of two experiments with similar results. (B) Serum HMGB1 ELISA after 6 h of reperfusion. Data are representative of two experiments with similar results. Gray, eritoran; white, control. *P < 0.05 when compared against control by one-way ANOVA. (C) Immunofluorescent stain of HMGB1 from sections of sham liver and liver 6 h after I/R in placebo control, and eritoran-treated mice (magnification 40×). Images are representative of liver sections from two mice per group. Red, HMGB1; blue, nuclei; green, F-actin.(D) TLR4 wild-type and knockout mice were given eritoran (5 mg/kg body weight) or vehicle control intraperitoneally 30 min before ischemia. HMGB1 levels in serum were collected 6 h after reperfusion and quantified using ELISA. Data represent the mean ± SEM (n ≥ 6 mice per group). Gray, eritoran; white, control. *P < 0.05, by one-way ANOVA.

DNA abnormalities are used in inclusion criteria of clinical trials for treatments with specific targeted molecules. MYC is one of the most powerful oncogenes and is known to be associated with triple-negative breast cancer (TNBC). Its DNA amplification is often part of the targeted DNA-sequencing panels under the assumption of reflecting upregulated signaling. However, it remains unclear if MYC DNA amplification is a surrogate of its upregulated signaling. Thus, we investigated the difference between MYC DNA amplification and mRNA high expression in TNBCs utilizing publicly available cohorts. MYC DNA amplified tumors were found to have various mRNA expression levels, suggesting that MYC DNA amplification does not always result in elevated MYC mRNA expression. Compared to other subtypes, both MYC DNA amplification and mRNA high expression were more frequent in the TNBCs. MYC mRNA high expression, but not DNA amplification, was significantly associated with worse overall survival in the TNBCs. The TNBCs with MYC mRNA high expression enriched MYC target genes, cell cycle related genes, and WNT/β-catenin gene sets, whereas none of them were enriched in MYC DNA amplified TNBCs. In conclusion, MYC mRNA high expression, but not DNA amplification, reflects not only its upregulated signaling pathway, but also clinical significance in TNBCs.

Solute carrier organic anion (SLCO) gene families encode organic anion transport proteins, which are transporters that up-take a number of substrates including androgens. Among them, high expression of SLCO2B1 is known to associate with the resistance to androgen deprivation therapy in prostate cancer (PCa). We hypothesized that high expression of SLCO genes enhances PCa progression by promoting the influx of androgen. Here, we demonstrated the impact of the expression levels of SLCO2B1 on prognosis in localized PCa after radical prostatectomy (RP) utilizing 494 PCa cases in The Cancer Genome Atlas (TCGA). SLCO2B1 high expression group showed significantly worse Disease-free survival (DFS) after RP (p = 0.001). The expression level of SLCO2B1 was significantly higher in advanced characteristics including Gleason Score (GS ≤ 6 vs GS = 7; p = 0.047, GS = 7 vs GS ≥ 8; p = 0.002), pathological primary tumor (pT2 vs pT3/4; p < 0.001), and surgical margin status (positive vs negative; p = 0.013), respectively. There was a significant difference in DFS between these two groups only in GS ≥ 8 patients (p = 0.006). Multivariate analysis demonstrated that only SLCO2B1 expression level was an independent predictor for DFS after RP in GS ≥ 8. SLCO2B1 high expressed tumors in GS ≥ 8 not only enriched epithelial mesenchymal transition (EMT) related gene set, (p = 0.027), as well as Hedgehog (p < 0.001), IL-6/JAK/STAT3 (p < 0.001), and K-ras signaling gene sets (p < 0.001), which are known to promote EMT, but also showed higher expression of EMT related genes, including N-cadherin (p = 0.024), SNAIL (p = 0.001), SLUG (p = 0.001), ZEB-1 (p < 0.001) and Vimentin (p < 0.001). In conclusion, PCa with high expression of SLCO2B1 demonstrated worse DFS, which might be due to accelerated EMT.

Lymphatic metastasis is known to contribute to worse prognosis of biliary tract cancer (BTC). Recently, sphingosine-1-phosphate (S1P), a bioactive lipid mediator generated by sphingosine kinase 1 (SPHK1), has been shown to play an important role in lymphangiogenesis and lymph node metastasis in several types of cancer. However, the role of the lipid mediator in BTC has never been examined. Here we found that S1P is elevated in BTC with the activation of ceramide-synthetic pathways, suggesting that BTC utilizes SPHK1 to promote lymphatic metastasis. We found that S1P, sphingosine and ceramide precursors such as monohexosyl-ceramide and sphingomyelin, but not ceramide, were significantly increased in BTC compared to normal biliary tract tissue using LC-ESI-MS/MS. Utilizing The Cancer Genome Atlas cohort, we demonstrated that S1P in BTC is generated via de novo pathway and exported via ABCC1. Further, we found that SPHK1 expression positively correlated with factors related to lymphatic metastasis in BTC. Finally, immunohistochemical examination revealed that gallbladder cancer with lymph node metastasis had significantly higher expression of phospho-SPHK1 than that without. Taken together, our data suggest that S1P generated in BTC contributes to lymphatic metastasis.

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An 82-year-old female presented to her breast surgeon with a hard, painful mass in the left breast. Mammography demonstrated a new hyperdense mass with pleomorphic calcifications of trabeculated appearance. Ultrasound-guided biopsy demonstrated a hypoechoic mass with significantly increased vascularity and tissue stiffness as well as additional irregular, hypoechoic masses in the same area. Together these findings suggested multifocal malignancy. The pathology report from the biopsy demonstrated fragments of solid sheets of epithelioid and focally spindled cells with multinucleated osteoclast giant cells. This was found to be most consistent with metaplastic carcinoma showing osteoblastic differentiation. The patient received a left-sided mastectomy. During follow-up with the patient, adjuvant chemotherapy was not advised given the relatively unknown survival advantage in this elderly patient.