Transcriptomic Profiles in Children With Septic Shock With or Without Immunoparalysis.

Background: Severe innate immune suppression, termed immunoparalysis, is associated with increased risks of nosocomial infection and mortality in children with septic shock. Currently, immunoparalysis cannot be clinically diagnosed in children, and mechanisms remain unclear. Transcriptomic studies identify subsets of septic children with downregulation of genes within adaptive immune pathways, but assays of immune function have not been performed as part of these studies, and little is known about transcriptomic profiles of children with immunoparalysis. Methods: We performed a nested case-control study to identify differences in RNA expression patterns between children with septic shock with immunoparalysis (defined as lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)α response < 200 pg/ml) vs those with normal LPS-induced TNFα response. Children were enrolled within 48 hours of the onset of septic shock and divided into two groups based on LPS-induced TNFα response. RNA was extracted from whole blood for RNAseq, differential expression analyses using DESeq2 software, and pathway analyses using Ingenuity Pathway Analysis. Results: 32 children were included in analyses. Comparing those with immunoparalysis (n =19) to those with normal TNFα response (n = 13), 2,303 transcripts were differentially expressed with absolute value fold change ≥ 1.5 and false discovery rate ≤ 0.05. The majority of downregulated pathways in children with immunoparalysis were pathways that involved interactions between innate and adaptive immune cells necessary for cell-mediated immunity, crosstalk between dendritic cells and natural killer cells, and natural killer cell signaling pathways. Upregulated pathways included those involved in humoral immunity (T helper cell type 2), corticotropin signaling, platelet activation (GP6 signaling), and leukocyte migration and extravasation. Conclusions: Our study suggests that gene expression data might be useful to identify children with immunoparalysis and identifies several key differentially regulated pathways involved in both innate and adaptive immunity. Our ongoing work in this area aims to dissect interactions between innate and adaptive immunity in septic children and to more fully elucidate patient-specific immunologic pathophysiology to guide individualized immunotherapeutic targets.

Assessment of Ki67 in Breast Cancer: Updated Recommendations From the International Ki67 in Breast Cancer Working Group.

Ki67 immunohistochemistry (IHC), commonly used as a proliferation marker in breast cancer, has limited value for treatment decisions due to questionable analytical validity. The International Ki67 in Breast Cancer Working Group (IKWG) consensus meeting, held in October 2019, assessed the current evidence for Ki67 IHC analytical validity and clinical utility in breast cancer, including the series of scoring studies the IKWG conducted on centrally stained tissues. Consensus observations and recommendations are: 1) as for estrogen receptor and HER2 testing, preanalytical handling considerations are critical; 2) a standardized visual scoring method has been established and is recommended for adoption; 3) participation in and evaluation of quality assurance and quality control programs is recommended to maintain analytical validity; and 4) the IKWG accepted that Ki67 IHC as a prognostic marker in breast cancer has clinical validity but concluded that clinical utility is evident only for prognosis estimation in anatomically favorable estrogen receptor-positive and HER2-negative patients to identify those who do not need adjuvant chemotherapy. In this T1-2, N0-1 patient group, the IKWG consensus is that Ki67 5% or less, or 30% or more, can be used to estimate prognosis. In conclusion, analytical validity of Ki67 IHC can be reached with careful attention to preanalytical issues and calibrated standardized visual scoring. Currently, clinical utility of Ki67 IHC in breast cancer care remains limited to prognosis assessment in stage I or II breast cancer. Further development of automated scoring might help to overcome some current limitations.

Obstacles, Opportunities and Priorities for Advancing Metastatic Breast Cancer Research.

In January 2016, the Metastatic Breast Cancer Alliance (the Alliance) convened a think tank of stakeholders from academia, government, industry, and patient advocacy to discuss gaps and opportunities in clinical and translational research in metastatic breast cancer. Priorities that emerged from the meeting included the following: the need for innovative preclinical model systems to study metastatic disease; increased sharing of resources and data; collaboration across cancer care teams and scientists; biorepositories for studies to identify biomarkers for treatment response; creation of patient registries to increase access to clinical trials and tissue procurement; and redesign of clinical trials in metastatic breast cancer. Cancer Res; 77(13); 3386-90. ©2017 AACR.

C6-ceramide and targeted inhibition of acid ceramidase induce synergistic decreases in breast cancer cell growth.

The sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death. Acid ceramidase (AC) hydrolyzes ceramide, and thus reduces intracellular levels of this proapoptotic lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in prostate cancer and in some breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosis-inducing effects of C6-ceramide (C6-cer) in breast cancer cells. Cultured breast cancer cells were treated with DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of reactive oxygen species, and mitochondrial transmembrane potential were determined. DM102 was a more effective AC inhibitor than N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(4'-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC(50) 5-10 µM) and DM102 (IC(50) 20 µM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index <0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of caspase 3 cleavage and a >3-fold increase in caspase 3/7 activation, PARP cleavage, and a >70% increase in Annexin-V positive cells. C6-cer/DM102 increased ROS levels 4-fold in MDA-MB-231 cells, shifted the ratio of Bax:Bcl-2 to >9-fold that of control cells, and resulted in mitochondrial membrane depolarization. DM102 also increased the synthesis of (3)H-palmitate-labeled long-chain ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/DM102 drug duo in human breast cancer.

Inhibition of acid ceramidase by a 2-substituted aminoethanol amide synergistically sensitizes prostate cancer cells to N-(4-hydroxyphenyl) retinamide.

BACKGROUND: The purpose of this study was to determine whether the therapeutic efficacy of fenretinide (4-HPR), a ceramide-generating anticancer agent, could be enhanced in prostate cancer cells by inclusion of a novel synthetic acid ceramidase (AC) inhibitor, DM102, a pivaloylamide of a 2-substituted aminoethanol. In prostate cancer, AC plays a role in progression and resistance to chemotherapy. METHODS: PC-3 and DU 145 hormone-refractory human prostate cancer cell lines were used. Cells were exposed to 4-HPR, DM102, and combinations; viability, apoptosis, cell migration, ceramide metabolism, and levels of reactive oxygen species (ROS) were assessed. RESULTS: Single agent 4-HPR and DM102 (2.5-10 µM) were weakly cytotoxic; however, combinations synergistically decreased cell viably to as low as 1.5% of control. N-oleoylethanolamine (NOE), a frequently employed AC inhibitor, was not effective in producing synergy. The 4-HPR/DM102 regimen enhanced caspase activity and increased [(3) H](dihydro)ceramide and ROS levels 6- and 30-fold over control, respectively. The antioxidant vitamin E, but not the de novo ceramide synthesis inhibitor myriocin, partially rescued cells from 4-HPR/DM102 cytotoxicity. The 4-HPR/DM102 combination also elicited synergistic cytotoxicity in DU 145 cells, another human hormone-refractory prostate cancer cell line. CONCLUSION: This study shows that 4-HPR cytotoxicity is enhanced in a synergistic fashion by inclusion of the AC inhibitor DM102, by a mechanism that enlists generation of ROS, and thus provides a system to raise 4-HPR therapeutic potential. The role of ceramide however in the cytotoxic response is not clear, as blocking ceramide generation failed to rescue PC-3 cells from 4-HPR/DM102 cytotoxicity.

Pilot study on the effects of dietary conjugated linoleic acid on tumorigenesis and gene expression in PyMT transgenic mice.

Conjugated linoleic acid (CLA) is a class of commercially available fatty acids that have been associated with anticancer properties in rodent models of chemical carcinogenesis. We conducted a pilot study to examine the antitumor effect of dietary CLA in a polyoma virus-middle T antigen (PyMT) mouse model of invasive breast cancer. Virgin 4-week-old PyMT mice were administered a mixed-isomer CLA diet (1% wt/wt) or control AIN-93G diet for 4 weeks (N = 6 and 5, respectively) and tumor burden was assessed at 8 weeks of age. Thoracic mammary glands were prepared as whole mounts with other glands being formalin fixed and paraffin embedded for histology and immunohistochemistry (IHC). Total RNA was prepared for microarray and real-time reverse transcription-polymerase chain reaction analysis. Western blots were performed for protein expression analysis. Tumor incidence was significantly increased in CLA-treated animals compared with controls (P = 0.009) and occurred with extensive lobular-alveolar expansion and loss of mammary adipose tissue. More than 100 genes were downregulated > or = 2-fold in the CLA-treated group compared with controls, including adipose-specific markers, as wells as cytoskeletal and adhesion-related genes. This was supported by dramatic decreases in the epithelial adherens E-cadherin and beta-catenin as demonstrated by IHC. Taken together, these results suggest that dietary CLA affects the mammary stromal environment, leading to tumor progression and cellular expansion in the PyMT mouse model. Further studies of the potential for cancer promotion are needed, especially because mixed-isomer CLA formulations are sold commercially as a nutritional supplement.

Metabolism of short-chain ceramide by human cancer cells--implications for therapeutic approaches.

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6muM) and high-dose (12muM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [(3)H]palmitic acid produced a 2-fold increase in (3)H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in (3)H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer.

t10c12 conjugated linoleic acid suppresses HER2 protein and enhances apoptosis in SKBr3 breast cancer cells: possible role of COX2.

BACKGROUND: HER2-targeted therapy with the monoclonal antibody trastuzumab (Herceptin) has improved disease-free survival for women diagnosed with HER2-positive breast cancers; however, treatment resistance and disease progression are not uncommon. Current data suggest that resistance to treatment in HER2 cancers may be a consequence of NF-kappaB overexpression and increased COX2-derived prostaglandin E2 (PGE(2)). Conjugated linoleic acid (CLA) has been shown to have anti-tumor properties and to inhibit NF-kappaB activity and COX2. METHODS: In this study, HER2-overexpressing SKBr3 breast cancer cells were treated with t10c12 CLA. Protein expression of the HER2 receptor, nuclear NF-kappaB p65, and total and phosphorylated IkappaB were examined by western blot and immunofluorescence. PGE(2) levels were determined by ELISA. Proliferation was measured by metabolism of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and apoptosis was measured by FITC-conjugated Annexin V staining and flow cytometry. RESULTS/CONCLUSIONS: We observed a significant decrease in HER2 protein expression on western blot following treatment with 40 and 80 microM t10c12 CLA (p<0.01 and 0.001, respectively) and loss of HER2 protein in cells using immunoflourescence that was most pronounced at 80 microM. Protein levels of nuclear NF-kappaB p65 were also significantly reduced at the 80 microM dose. This was accompanied by a significant decrease in PGE(2) levels (p = 0.05). Pretreatment with t10c12 CLA significantly enhanced TNFalpha-induced apoptosis and the anti-proliferative action of trastuzumab (p = 0.05 and 0.001, respectively). These data add to previous reports of an anti-tumor effect of t10c12 CLA and suggest an effect on the HER2 oncogene that may be through CLA mediated downregulation of COX2-derived PGE(2).