Increased apoptosis is associated with robust immune cell infiltration and cytolytic activity in breast cancer

Tumor infiltrating immune cells plays a critical role in cancer progression. Apoptosis is an autonomous cell death that counteracts tumor growth. To this end, we hypothesized that increased apoptosis in breast cancer is associated with immune cell killing. Apoptosis score of MSigDB Hallmark collection was used to analyze METABRIC cohort (n=1904) and TCGA (n=1069) as validation cohort. High apoptosis tumors enriched cancer promoting signaling pathways; hypoxia, KRAS, TGF-β, PI3K signaling, and was associated with low MKI67 expression and less cell proliferation gene sets, less homologous recombination defects, and less altered fraction. High apoptosis tumors also enriched angiogenesis and high infiltration of vascular endothelial cells, pericytes and stromal cells and significantly enriched inflammation and immune response-related gene sets and high infiltration of CD8, CD4 memory, dendritic cells, M1 and M2 macrophages and significant elevation of cytolytic activity and immune checkpoint molecules, consistently in both cohorts. In conclusion, breast cancer patients with high apoptosis are associated with angiogenesis, immune response, high immune cell infiltration and cytolytic activity. To the best of our knowledge, this is the first study to utilize in silico translational approach to demonstrate the clinical relevance of apoptosis in breast cancer patients in large cohorts.     

Hyaluronan metabolism enhanced during epidermal differentiation is suppressed by vitamin C

Epidermis, the surface layer of human skin, is formed by cells called keratinocytes. They divide (making more cells) in the deeper parts of the skin and gradually move towards the surface. Keratinocytes eventually form a layer of dead cells on the skin's surface, and this layer is the main protective barrier against things that might damage or irritate the skin, such as chemicals and germs. A viscous substance called hyaluronan surrounds keratinocytes in the lower, living cell layers. All kinds of injuries to the epidermis, like wounding, infection, and sunburn, trigger a strong inflammatory response by keratinocytes. This response includes increased division of keratinocytes, and their movement to help the injury site recover, as well as increased hyaluronan production and turnover. In many skin diseases the keratinocyte injury‐response is excessive, or even completely unfounded, and harmful. Therefore, factors and treatments that calm down this reaction are potential medicines for some skin diseases. This study from Kuopio, Finland, studied hyaluronan metabolism (i.e. breakdown) in a special model developed in the laboratory, in which keratinocytes separate to produce a normal‐looking epidermis. This model allowed the researchers to demonstrate that vitamin C suppresses both production and degradation of hyaluronan in the epidermis. Since rapid hyaluronan metabolism is an indicator of inflammation, vitamin C – which is an antioxidant ‐ might have a use for suppressing this reaction in keratinocytes. This study calls for more work on the vitamin's mechanism of action, and possible roles in clinical practice.     

Circulating interleukin-1β and interleukin-6 concentrations are closely associated with γ-glutamyltranspeptidase activity in middle-aged Japanese men without obvious cardiovascular diseases

Interleukin (IL)-1β and IL-6 expressions are known to be induced by oxidant stress. In the present study, we examined the relationships between these interleukins and the activity of γ-glutamyltranspeptidase (γ-GTP), which was recently reported as a source of oxidant stress production, in the circulating blood of middle-aged Japanese men without obvious cardiovascular diseases. We conducted a cross-sectional study of 317 Japanese men without obvious cardiovascular diseases aged 40 to 69 years (mean ± SD, 58.6 ± 7.6 years) who participated in health checkups in Japan. We analyzed their clinical parameters in serum, lifestyle factors, and plasma IL-1β and IL-6 concentrations. We compared the relationships between these interleukin concentrations and the clinical parameters and lifestyle factors by Spearman correlation coefficients. Stepwise multiple linear regression analyses for interleukins based on the other parameters and γ-GTP, which were classified into 3 groups according to the concentrations, were performed. Interleukin-1β and IL-6 concentrations were closely associated with γ-GTP activity but less associated with alanine aminotransferase and aspartate aminotransferase activities by Spearman correlation coefficients. Stepwise multiple linear regression analyses showed that γ-GTP activity was the explanatory variable for elevated IL-1β and IL-6 concentrations. As natural logarithms, the IL-1β and IL-6 concentrations were estimated to be 1.734- and 1.157-fold higher, respectively, in subjects with high γ-GTP activity ranges than in subjects with a low γ-GTP activity range. The present results show that circulating IL-1β and IL-6 concentrations are strongly and independently associated with γ-GTP activity in middle-aged Japanese men without obvious cardiovascular diseases.     

MELK expression in breast cancer is associated with infiltration of immune cell and pathological compete response (pCR) after neoadjuvant chemotherapy

In experimental settings, maternal embryonic leucine zipper kinase (MELK), an apical member of the snf1/AMPK serine-threonine kinases family, plays a role in tumor growth. We investigated the clinical relevance of MELK expression by performing silico analyses of 7,135 breast cancer patients using multiple independent large cohorts. In triple negative breast cancer (TNBC) found that elevated MELK expression significantly correlates with Nottingham histologic grade and tumor growth according to American Joint Committee Cancer (AJCC) stage. High MELK tumor enriched cell proliferation-related gene sets as well as DNA repair, unfolded protein response, and MTORC signaling gene sets. In two independent cohorts a high mutation rate and worse survival was significantly associated with high MELK tumor. In immune-related gene sets including, allograft rejection, interferon (IFN)-α response, and IFN-γ response, high MELK tumor significantly enriched. Pro-cancer regulatory T cells, T helper type 2 cells and anti-cancer immune cells including CD4⁺ memory T cells, T helper type1 cells, CD8⁺ T cells, M1 macrophages, gamma-delta T cells, and dendritic cells with high levels of cytolytic activity (CYT) were highly infiltrated. MELK expression did not correlate with the responses to any of the drugs tested in cell lines. However, pathologic complete response was significantly associated with high MELK following NAC in both TNBC and ER-positive plus HER2-negative breast cancer. In conclusion, cell proliferation, immune response, and NAC breast cancer response was associated with MELK expression.     

Cross-talk between LPA1 and epidermal growth factor receptors mediates up-regulation of sphingosine kinase 1 to promote gastric cancer cell motility and invasion

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA(1) receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(1) receptor. An LPA(1) receptor antagonist or down-regulation of its expression prevented SphK1 and S1P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P(3) expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P(3), but not S1P(1), also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.     

Minimum sizes for remnant and transplanted livers in rats

Our new technique of hepatectomy in rats, avoiding inferior vena cava constriction, revealed that 5%–10% of the total liver is the minimum required volume for remnant liver, a smaller amount than previously reported. The characteristic histological findings of remnant liver in perioperative death are marked hepatocytosis and hepatocyte apoptosis, and extensive hepatocyte necrosis with sinusoid congestion, all of which are thought to be caused by hyperendotoxemia and mitochondrial energy crisis. Partial liver transplantation in rats with our new technique of hepatectomy in the donor operation showed that a graft of 20% liver was the minimum required volume for successful transplantation, also a smaller amount than previously reported. There was no difference in the rate of increase in wet weight after surgery between 20% remnant liver and 20% transplanted liver. Differences between 10% minimum volume remnant liver and 20% minimum transplanted liver appeared to be caused by ischemia-reperfusion and cold preservation injury to the transplanted liver.     

Cryoprotective effects of various saccharides on cryopreserved mouse sperm from various strains

Aim  Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm. Methods  Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4–33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization. Results  The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion  Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm.     

Interstitial Fluid Sphingosine-1-Phosphate in Murine Mammary Gland and Cancer and Human Breast Tissue and Cancer Determined by Novel Methods

The tumor microenvironment is a determining factor for cancer biology and progression. Sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphKs), is a bioactive lipid mediator that regulates processes important for cancer progression. Despite its critical roles, the levels of S1P in interstitial fluid (IF), an important component of the tumor microenvironment, have never previously been measured due to a lack of efficient methods for collecting and quantifying IF. The purpose of this study is to clarify the levels of S1P in the IF from murine mammary glands and its tumors utilizing our novel methods. We developed an improved centrifugation method to collect IF. Sphingolipids in IF, blood, and tissue samples were measured by mass spectrometry. In mice with a deletion of SphK1, but not SphK2, levels of S1P in IF from the mammary glands were greatly attenuated. Levels of S1P in IF from mammary tumors were reduced when tumor growth was suppressed by oral administration of FTY720/fingolimod. Importantly, sphingosine, dihydro-sphingosine, and S1P levels, but not dihydro-S1P, were significantly higher in human breast tumor tissue IF than in the normal breast tissue IF. To our knowledge, this is the first reported S1P IF measurement in murine normal mammary glands and mammary tumors, as well as in human patients with breast cancer. S1P tumor IF measurement illuminates new aspects of the role of S1P in the tumor microenvironment.