Experimental and Computational Studies Reveal Novel Interaction of Lymphocytes Antigen 6K to TGF-ß Receptor Complex.

TGF-ß signaling promotes migration, invasion, and distant colonization of cancer cells in advanced metastatic cancers. TGF-ß signaling suppresses the anti-tumor immune response in a tumor microenvironment, allowing sustained tumor growth. TGF-ß plays an important role in normal physiology; thus it is no surprise that the clinical development of effective and safe TGF-ß inhibitors has been hampered due to their high toxicity. We discovered that increased expression of LY6K in cancer cells led to increased TGF-ß signaling and that inhibition of LY6K could lead to reduced TGF-ß signaling and reduced in vivo tumor growth. LY6K is a highly cancer-specific protein, and it is not expressed in normal organs except in the testes. Thus, LY6K is a valid target for developing therapeutic strategies to inhibit TGF-ß signaling in cancer cells. We employed in vitro pull-down assays and molecular dynamics simulations to understand the structural determinants of the TGF-ß receptor complex with LY6K. This combined approach allowed us to identify the critical residues and dynamics of the LY6K interaction with the TGF-ß receptor complex. These data are critical in designing novel drugs for the inhibition of TGF-ß in LY6K expressing cancer, induction of anti-tumor immune response, and inhibition of tumor growth and metastatic spread.

M2-like macrophages induce colon cancer cell invasion via matrix metalloproteinases.

The inflammatory milieu plays an important role in colon cancer development and progression. Previously, we have shown that tumor-associated macrophages (TAMs), an important component of the tumor microenvironment, are enriched in tumors compared with normal tissue and confer a poorer prognosis. In the present study, we found that matrix metallopeptidase-9 (MMP-9), which degrades extracellular matrix proteins, was increased in biopsies from colon cancer patients and in mouse xenografts with SW480 cell-derived tumors. SW480 colon cancer cells exposed to M2-like macrophage-conditioned medium (M2-medium) exhibited increased MMP-9 mRNA, protein expression and gelatinase activity. A similar effect was obtained by the addition of tumor necrosis factor-α (TNFα) and leukotriene D4 (LTD4 ). MMP-9 expression and activity were reduced by a TNFα blocking antibody adalimumab and a cysteinyl leukotriene receptor 1 (CysLTR1, the receptor for LTD4 ) antagonist montelukast. M2-medium also induced changes in the epithelial-mesenchymal transition (EMT) markers E-cadherin, ß-catenin, vimentin, and snail in SW480 cells. We also found that both M2-medium and TNFα and LTD4 induced stabilization/nuclear translocation of ß-catenin. Furthermore, we also observed an elongated phenotype that may indicate increased invasiveness, as confirmed in a collagen I invasion assay. M2-medium increased the invasive ability, and a similar effect was also obtained by the addition of TNFα and LTD4 . The specific MMP inhibitor I or adalimumab and montelukast reduced the number of invasive cells. In conclusion, our findings show that M2-medium enriched in TNFα and LTD4 promote colon cancer cell invasion via MMP-9 expression and activation and the induction of EMT.

The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model.

BACKGROUND: Colorectal cancer is one of the most common types of cancers worldwide. Recent studies have identified cancer-initiating cells (CICs) as a subgroup of replication-competent cells in the development of colorectal cancer. Although it is understood that an inflammation-rich tumor microenvironment presumably supports CIC functions, the contributory factors are not very well defined. The present study advances our understanding of the role of the eicosanoids leukotriene D4 (LTD4) and prostaglandin E2 (PGE2) in the tumorigenic ability of CICs and investigates the consequential changes occurring in the tumor environment that might support tumor growth. METHODS: In this study we used human HCT-116 colon cancer ALDH(+) cells in a nude mouse xenograft model. Protein expression and immune cell was determined in tumor-dispersed cells by flow cytometry and in tumor sections by immunohistochemistry. mRNA expressions were quantified using RT-q-PCR and plasma cytokine levels by Multiplex ELISA. RESULTS: We observed that LTD4 and PGE2 treatment augmented CIC-induced tumor growth. LTD4-and PGE2-treated xenograft tumors revealed a robust increase in ALDH and Dclk1 protein expression, coupled with activated ß-catenin signaling and COX-2 up-regulation. Furthermore, LTD4 or PGE2 accentuated the accumulation of CD45 expressing cells within xenograft tumors. Further analysis revealed that these infiltrating immune cells consisted of neutrophils (LY6G) and M2 type macrophages (CD206(+)). In addition, LTD4 and PGE2 treatment significantly elevated the plasma levels of cysteinyl leukotrienes and PGE2, as well as levels of IL-1ß, IL-2, IL-6, TNF-α and CXCL1/KC/GRO. In addition, increased mRNA expression of IL-1ß, IL-6 and IL-10 were detected in tumors from mice that had been treated with LTD4 or PGE2. CONCLUSION: Our data suggest that both LTD4 and PGE2 promote CICs in initiating tumor growth by allowing modifications in the tumor environment. Our data indicate that new therapeutic strategies targeting eicosanoids, specifically LTD4 and PGE2, could be tested for better therapeutic management of colon cancer.

NSC243928 Treatment Induces Anti-Tumor Immune Response in Mouse Mammary Tumor Models.

NSC243928 induces cell death in triple-negative breast cancer cells in a LY6K-dependent manner. NSC243928 has been reported as an anti-cancer agent in the NCI small molecule library. The molecular mechanism of NSC243928 as an anti-cancer agent in the treatment of tumor growth in the syngeneic mouse model has not been established. With the success of immunotherapies, novel anti-cancer drugs that may elicit an anti-tumor immune response are of high interest in the development of novel drugs to treat solid cancer. Thus, we focused on studying whether NSC243928 may elicit an anti-tumor immune response in the in vivo mammary tumor models of 4T1 and E0771. We observed that NSC243928 induced immunogenic cell death in 4T1 and E0771 cells. Furthermore, NSC243928 mounted an anti-tumor immune response by increasing immune cells such as patrolling monocytes, NKT cells, B1 cells, and decreasing PMN MDSCs in vivo. Further studies are required to understand the exact mechanism of NSC243928 action in inducing an anti-tumor immune response in vivo, which can be used to determine a molecular signature associated with NSC243928 efficacy. NSC243928 may be a good target for future immuno-oncology drug development for breast cancer.

Lymphocyte antigen 6K signaling to aurora kinase promotes advancement of the cell cycle and the growth of cancer cells, which is inhibited by LY6K-NSC243928 interaction.

Lymphocyte antigen 6K (LY6K) is a small GPI-linked protein that is normally expressed in testes. Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including cancers of the breast, ovary, gastrointestinal tract, head and neck, brain, bladder, and lung. LY6K is required for ERK-AKT and TGF-ß pathways in cancer cells and is required for in vivo tumor growth. In this report, we describe a novel role for LY6K in mitosis and cytokinesis through aurora B kinase and its substrate histone H3 signaling axis. Further, we describe the structural basis of the molecular interaction of small molecule NSC243928 with LY6K protein and the disruption of LY6K-aurora B signaling in cell cycle progression due to LY6K-NSC243928 interaction. Overall, disruption of LY6K function via NSC243928 led to failed cytokinesis, multinucleated cells, DNA damage, senescence, and apoptosis of cancer cells. LY6K is not required for vital organ function, thus inhibition of LY6K signaling is an ideal therapeutic approach for hard-to-treat cancers that lack targeted therapy such as triple-negative breast cancer.

Listeria delivers tetanus toxoid protein to pancreatic tumors and induces cancer cell death in mice.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT856-1313) into PDAC tumor cells by attenuated Listeria monocytogenes. This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria-TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria-TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria-TT + GEM-treated mice demonstrated a CD4 T cell-mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node-like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria-TT or Listeria-TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria-TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria-delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.

Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice.

BACKGROUND: Treatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor's immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B3), in mice with pancreatic cancer. METHODS: Various mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (KrasG12D, p53R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope. RESULTS: A significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading. CONCLUSION: This study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

Montelukast, a CysLT1 receptor antagonist, reduces colon cancer stemness and tumor burden in a mouse xenograft model of human colon cancer.

Inflammation is implicated in the etiology of sporadic colon cancer (CC), which is one of the leading causes of cancer-related deaths worldwide. Here, we report that inhibition of the inflammatory receptor CysLT1 through its antagonist, montelukast, is beneficial in minimizing stemness in CC and thereby minimizing tumor growth in a mouse xenograft model of human colon cancer. Upon treatment with montelukast, colonospheres derived from HT-29 and SW-480 human colon cancer cells exhibited a significant phenotypic change coupled with the downregulation of mRNA and protein expression of cancer stem cell (CSC) markers ALDH1 and DCLK1. Moreover, montelukast reduced the size of HT-29 cell-derived tumors in mice. The reduction in tumor size was associated with decreased levels of ALDH1A1, DCLK1, BCL2 mRNA and macrophage infiltration into the tumor tissue. Interestingly, this treatment elevated levels of the tumor suppressor 15-PGDH while reducing COX-2 expression. Our data highlight the association of CysLT1R with CSCs and demonstrate that inhibition of CysLT1R could prove beneficial in minimizing CSC-induced tumor growth. This work advances the notion that targeting CSCs is a promising approach to improve outcomes in those afflicted with colon cancer.

Immunotherapy with Listeria reduces metastatic breast cancer in young and old mice through different mechanisms.

Cancer immunotherapy is one of the most promising and benign therapies against metastatic cancer. However, most cancer patients are old and elderly react less efficient to cancer vaccines than young adults, due to T cell unresponsiveness. Here we present data of cancer vaccination in young and old mice with metastatic breast cancer (4T1 model). We tested adaptive and innate immune responses to foreign antigens (Listeria-derived) and self-antigens (tumor-associated antigens (TAA)) and their contribution to elimination of metastases at young and old age. Three different protocols were tested with Listeria: a semi- and exclusive-therapeutic protocol both one-week apart, and an exclusive therapeutic protocol frequently administered. Adaptive and innate immune responses were measured by ELISPOT in correlation with efficacy in the 4T1 model. We found that Listeria induced immunogenic tumor cell death, resulting in CD8 T cell responses to multiple TAA expressed by the 4T1 tumors. Only exclusive therapeutic frequent immunizations were able to overcome immune suppression and to activate TAA- and Listeria-specific CD8 T cells, in correlation with a strong reduction in metastases at both ages. However, MHC class Ia antibodies showed inhibition of CD8 T cell responses to TAA at young but not at old age, and CD8 T cell depletions in vivo demonstrated that the T cells contributed to reduction in metastases at young age only. These results indicate that CD8 T cells activated by Listeria has an antitumor effect at young but not at old age, and that metastases at old age have been eliminated through different mechanism(s).

32-Phosphorus selectively delivered by listeria to pancreatic cancer demonstrates a strong therapeutic effect.

Our laboratory has developed a novel delivery platform using an attenuated non-toxic and non-pathogenic bacterium Listeria monocytogenes that infects tumor cells and selectively survives and multiplies in metastases and primary tumors with help of myeloid-derived suppressor cells (MDSC) and immune suppression in the tumor microenvironment (TME). 32P was efficiently incorporated into the Listeria bacteria by starvation of the bacteria in saline, and then cultured in phosphorus-free medium complemented with 32P as a nutrient. Listeria-32P kills tumor cells through both 32P-induced ionizing radiation and Listeria-induced reactive oxygen species (ROS). The levels of 32P and Listeria were studied in various normal and tumor tissues, at sequential time points after injection of mice with pancreatic cancer (syngeneic model Panc-02). We found that 32P and Listeria predominantly accumulated in tumors and metastases, with their highest accumulation 4 hrs (32P) and 3 days (Listeria) after injection. Listeria also penetrated the transgenic KPC (conditionally express endogenous Kras-G12D and p53-R172H mutant alleles) pancreatic tumors and metastases. This is remarkable since KPC tumors, like human tumors, exhibit a stromal barrier, which prevents most drugs from penetrating the pancreatic tumors. Therapeutic treatment with Listeria -32P resulted in a strong reduction of the growth of pancreatic cancer at early and late stages in Panc-02 and KPC mice. These results highlight the power of Listeria as new delivery platform of anticancer agents to the TME. Not only were therapeutic levels of radioactive Listeria reached in tumors and metastases but the selective delivery also led to minimal side effects.

Lactational Exposure to Di (2-ethylhexyl) Phthalate Impairs the Ovarian and Uterine Function of Adult Offspring Rat.

Phthalates, a class of chemicals used as plasticizers, are economically important due to several industrial applications. Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used phthalate plasticizer, and it has been described as a potent antiandrogen in males. In this study, lactating dams were exposed via oral gavage to corn oil (vehicle) and DEHP (1, 10, and 100 mg/kg body weight) from postnatal day 1 to 21, and the effects were evaluated in the ovary and uterus of F(1) progeny. DEHP exposure significantly decreased the body weight and organ weight in a dose-dependent manner. Serum levels of estradiol, testosterone, and progesterone were decreased but anogenital distance was unaffected. The mRNA expressions of luteinizing hormone receptor, follicle-stimulating hormone receptor, androgen receptor, estrogen receptor (ERα and ERß), progesterone receptor, peroxisome proliferator-activated receptor γ, 3ß hydroxysteroid dehydrogenase, aromatase, and steroidogenic acute regulatory protein were altered in the ovary of F1 progeny rats. Our finding suggest that lactational exposure to DEHP has transgenerational effect on female reproductive system.

Broad spectrum antimicrobial compounds from the bacterium Exiguobacterium mexicanum MSSRFS9.

Clinical bacterial pathogens front a major challenge for the clinical researchers and physicians. In particular microbial pathogens like Escherichia coli, Shigella flexneri, Klebsiella pneumonia and Salmonella enterica are apparelled with systemic machineries to bring down the human immune system as well as proliferate dramatically in a short period which in turn cause a pronounced ailment to the human health. In vitro evaluation of four purified compounds isolated from rhizosphere bacterium Exiguobacterium mexicanum tested against clinical pathogens mentioned above by disc diffusion method showed the two compounds viz., 3,6,18-trione, 9,10-dihydro-12'-hydroxyl-2methyl-5-(phenyl methyl) (5'-alpha, 10-alpha)-dihydroergotamine (C3) and dipropyl - S-propyl ester (C4) exhibit antibacterial property against all the tested pathogens. Among the four clinical pathogens tested, compound C3 has shown higher zone of inhibition against S. enterica with 17±0 mm, followed by S. flexneri with 16.5±0.7 mm, E. coli with 15±0 mm and K. pneumoniae with 14±0 mm, respectively. The compound C4 has shown higher antimicrobial activity against S. enterica with 21.5±0.7 mm zone of inhibition, followed by S. flexneri with 19.5±0.7 mm, E. coli with 17±0 mm and K. pneumoniae with 16±0 mm, these two compounds were found to be safer when subjected to rat haematological and enzymatic analysis.

Apoptosis induction by an analog of curcumin (BDMC-A) in human laryngeal carcinoma cells through intrinsic and extrinsic pathways.

BACKGROUND: Head and neck cancer is the sixth most frequently occurring cancer worldwide and accounts for about 2% of all cancer-related deaths annually. Curcumin is a well-known chemopreventive agent, and apoptosis induction by curcumin has been reported in many cancer cell types. We synthesized an ortho-hydroxy substituted analog of curcumin, bisdemethoxycurcumin analog (BDMC-A), and aimed to demarcate the apoptotic effects induced by BDMC-A on human laryngeal cancer Hep-2 cells and to compare these effects with those induced by curcumin. METHODS: We evaluated the apoptotic effects of BDMC-A in comparison to those of curcumin on Hep-2 cells by performing Western blotting, RT-PCR, fluorescent staining and DNA fragmentation assays. In addition, we carried out an in silico molecular docking study on the EGFR kinase domain. RESULTS: We found that BDMC-A can induce apoptosis in Hep-2 cells by regulating the expression of both intrinsic and extrinsic apoptotic proteins, i.e., Bcl-2, Bax, apoptososme complex and death receptors, more efficiently than curcumin. We also observed increased nuclear fragmentation and chromatin condensation after BDMC-A treatment compared to curcumin treatment. Depolarized mitochondria and ROS generation was well pronounced in both BDMC-A and curcumin treated Hep-2 cells. Our in silico molecular docking study on the EGFR kinase domain revealed that BDMC-A may dock more efficiently than curcumin. CONCLUSIONS: From our results we conclude that BDMC-A can induce apoptosis in Hep-2 laryngeal carcinoma cells more effectively than curcumin, and that this activity can be attributed to the presence of a hydroxyl group at the ortho position within this compound.