Development of fluorophore labeled or biotinylated anticancer small molecule NSC243928.

Small molecule NSC243928 binds with LY6K, a potential target for the treatment of triple-negative breast cancer, and induces cancer cell death with an unclear mechanism. We have developed chemical tools to identify the molecular mechanisms of NSC243928-LY6K interaction. Herein, we report on the development and synthesis of biotinylated and fluorophore-tethered derivatives of NSC243928 guided by docking studies and molecular dynamics. Surface plasmon resonance assay indicates that these derivatives retained a direct binding with LY6K protein. Confocal analysis revealed that nitrobenzoxadiazole (NBD) fluorophore tagged NSC243928 is retained in LY6K expressing cancer cells. These novel modified compounds will be employed in future in vitro and in vivo studies to understand the molecular mechanisms of NSC243928 mediated cancer cell death. These studies will pave the path for developing novel targeted therapeutics and understanding any potential side-effects of these treatments for hard-to-treat cancers such as triple-negative breast cancer or other cancers with high expression of LY6K.

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.

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.

Current concepts in the evaluation and treatment of patients with diffuse malignant peritoneal mesothelioma.

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and ultimately fatal cancer that was first recognized and described a century ago. It is a diffuse primary malignant condition arising from the mesothelial lining of the peritoneum, and its natural history is hallmarked by a propensity to progress almost exclusively within the abdominal cavity throughout the entire course of disease. Patients afflicted with DMPM most commonly present with nonspecific abdominal symptoms that lead to diagnosis when the condition is relatively advanced. Historically, median overall survival for patients with DMPM without treatment is very short, averaging 6 months. Systemic chemotherapy using pemetrexed and cisplatin has an overall response rate of approximately 25% and a median overall survival of approximately 1 year. Many institutional reports have shown that in selected patients, operative cytoreduction and hyperthermic intraoperative peritoneal chemotherapy using cisplatin or mitomycin C is associated with long-term survival. Recent studies on the molecular biology of DMPM have yielded new insights relating to the potentially important role of the phosphatidylinositol 3-kinase/mammalian target of rapamycin and epidermal growth factor receptor pathways in this disease, which may translate into new therapeutic options for patients with DMPM.

Surgery for peritoneal mesothelioma.

Malignant peritoneal mesothelioma (MPM) is a rare, progressive, and ultimately fatal disease in almost all afflicted individuals. MPM is a cancer that arises diffusely from the serosa of the abdominal cavity and progresses almost exclusively in this region; understanding its characteristic tumor biology has been the reasoning behind the development of treatment strategies designed to control disease progression in the peritoneum. This principally includes operative cytoreduction and hyperthermic intra-operative perfusion of intraperitoneal chemotherapy (HIPEC). This approach has been administered either alone or in combination with early postoperative intraperitoneal chemotherapy (EPIC), or as a component of a more protracted multimodal approach employing initial debulking surgery, intraperitoneal chemotherapy, and whole abdominal radiotherapy. Median overall survivals of up to 7 years have been observed in series of patients selected for operative cytoreduction and HIPEC. Factors associated with good outcome are female gender, age less or equal to 60 years, and the ability to achieve a complete extirpation of all gross peritoneal disease. In patients with symptomatic ascites, complete palliation is achieved in almost all cases. However, this treatment strategy is not without complications and carries a morbidity of 25% and mortality up to 7%. Despite these risks, the best overall survival data have been associated with this surgical approach. At our institution, we advocate cytoreduction and HIPEC as the standard management for patients with MPM for whom operative cytoreduction appears possible and safe. We believe this treatment approach should be considered as the standard of care for patients with MPM.

Isolated hepatic perfusion with high-dose melphalan results in immediate alterations in tumor gene expression in patients with metastatic ocular melanoma.

BACKGROUND: Patients with ocular melanoma liver metastases have a poor prognosis, treatment options are limited, and median survival is less than 1 year. In this study, we characterized the early molecular changes that occur in tumors immediately after vascular isolation perfusion with melphalan with hyperthermia in patients with hepatic metastases from ocular melanoma. METHODS: Patients underwent treatment on a clinical trial using a 60-min hyperthermic isolated hepatic perfusion (IHP) with melphalan. Microarray analysis was performed in 28 tumor samples obtained intraoperatively of which 12 were pre- and 16 were post-IHP. Various statistical analyses were performed to identify differentially expressed genes and gene categories between the groups. RESULTS: Median survival of 17 treated patients was 11.9 months. Unsupervised hierarchical clustering of all tumors resulted in separation of pre and post-IHP samples into two distinct groups. Analysis of genes showed that the Ras GTPase activator, ecotropic viral integration site 5 (EVI5), and several other melanoma-associated genes were overexpressed in pre-IHP tumors. In post-IHP samples the overexpression of a DNA replication associated gene, replication factor C (RFC5), was significantly associated with shortened survival (P < 0.003). Other major gene ontology categories identified in the post-IHP tumor samples were DNA-directed RNA polymerase activity and chromatin remodeling, both important categories involved in DNA replication and repair. CONCLUSIONS: These results demonstrate that acute changes in gene expression patterns occur in tumors immediately after treatment with melphalan administered via hyperthermic IHP. Rapid activation of DNA synthesis and repair pathways may be a mechanism of acquired tumor resistance in patients with ocular melanoma.

Site-specific gene expression profiles and novel molecular prognostic factors in patients with lower gastrointestinal adenocarcinoma diffusely metastatic to liver or peritoneum.

BACKGROUND: Generally, colorectal and high-grade appendiceal cancers are treated similarly; treatment approach is primarily based on tumor histology and stage of disease. Patients with adenocarcinoma of the lower gastrointestinal tract frequently experience diffuse metastases isolated to liver or peritoneum and have a poor survival. Identification of novel molecular pathways in metastases from these patients may identify novel targets and prognostic factors. METHODS: Microarray analyses of 20 metastatic tumors from patients with colorectal adenocarcinoma isolated to liver or peritoneum and eight high-grade appendiceal adenocarcinoma metastatic to peritoneum were performed using oligonucleotide microarray. RESULTS: In an unsupervised hierarchical cluster analysis of 2-fold upregulated or downregulated genes, there was a clear site-specific segregation of liver versus peritoneal metastases. Genes primarily involved in metastasis, angiogenesis, cell cycle regulation, cell proliferation, and cell adhesion were distinctly altered between these two metastatic sites. Among the metastasis genes, the average expression levels of LI-cadherin, ALCAM, CD2, and CD14 were significantly higher in both metastatic sites. TIMP1 was overexpressed in both sites where as TIMP-2, IGF-1, and HIF-1alpha were upregulated only in peritoneal metastases demonstrating the potential benefit of metastasis site-specific treatments. Subsets of genes significantly associated with poor survival were defined, a RET proto-oncogene interacting gene, GOLGA5, was highly predictive for survival in patients with colorectal adenocarcinoma. CONCLUSIONS: These results demonstrate that liver and peritoneal metastases of lower GI adenocarcinoma have distinct gene expression patterns; these distinctions may help in the development of therapies based on site of metastases.

The role of interleukin 1 in growth and metastasis of human cancer xenografts.

BACKGROUND: Interleukin 1 (IL-1) is a pluripotent cytokine that promotes angiogenesis, tumor growth, and metastasis in experimental models; its presence in some human cancers is associated with aggressive tumor biology. The purpose of these studies was to characterize the role of IL-1 in human cancers and determine if inhibition of IL-1 via its receptor antagonist, IL-1Ra, alters tumor growth and metastatic potential. METHODS: IL-1 mRNA or protein levels were determined in clinical tumor samples, cancer cell lines, and xenografts using quantitative reverse transcription-PCR or ELISA. Biological activity of tumor-derived IL-1 protein was shown via induction of permeability across endothelial cell monolayers. The effects of recombinant IL-1Ra on tumor lines in culture (cell proliferation and IL-8 secretion) and in xenograft models (tumor growth, metastatic potential, and intratumoral levels of IL-8 and VEGF) were characterized. The effects of IL-1Ra-mediated regression of xenograft growth on angiogenic proteins (IL-8 and VEGF) were evaluated in an IL-1-producing melanoma (SMEL) xenograft model. RESULTS: IL-1 mRNA was highly expressed in more than half of all tested metastatic human tumor specimens including non-small-cell lung carcinoma, colorectal adenocarcinoma, and melanoma tumor samples. Constitutive IL-1 mRNA expression was identified in several cancer cell lines; tumor supernatant from these cell lines produced a significant increase in endothelial cell monolayer permeability, a hallmark event in early angiogenesis, in an IL-1-dependent manner. Moreover, systemic recombinant IL-1Ra resulted in significant inhibition of xenograft growth and neovessel density of IL-1-producing, but not non-IL-1-producing, tumor cell lines. Subsequent analysis of SMEL, a melanoma cell line with constitutive IL-1 production, showed that neither exogenous IL-1 nor IL-1Ra altered tumor cell proliferation rates in vitro. Gene expression analyses of IL-1Ra-treated SMEL xenografts showed a >3-fold down-regulation of 100 genes compared with control including a marked down-regulation of IL-8 and VEGF. CONCLUSIONS: These data show that the IL-1 gene is frequently expressed in metastases from patients with several types of human cancers. IL-1Ra inhibits xenograft growth in IL-1-producing tumors but has no direct antiproliferative effects in vitro; decreased tumor levels of IL-8 and VEGF may be an early surrogate of IL-1Ra-mediated antitumor activity. IL-1Ra may have a role alone or with other agents in the treatment of human cancers.

Interleukin-1 and cancer progression: the emerging role of interleukin-1 receptor antagonist as a novel therapeutic agent in cancer treatment.

The tumor microenvironment consists of tumor, immune, stromal, and inflammatory cells which produce cytokines, growth factors, and adhesion molecules that promote tumor progression and metastasis. Of particular interest in this setting is interleukin-1 (IL-1), a pleiotropic cytokine with numerous roles in both physiological and pathological states. It is known to be up regulated in many tumor types and has been implicated as a factor in tumor progression via the expression of metastatic and angiogenic genes and growth factors. A number of studies have reported that high IL-1 concentrations within the tumor microenvironment are associated with a more virulent tumor phenotype. Solid tumors in which IL-1 has been shown to be up regulated include breast, colon, lung, head and neck cancers, and melanomas, and patients with IL-1 producing tumors have generally bad prognoses. The exact mechanisms by which IL-1 promotes tumor growth remain unclear, though the protein is believed to act via induction of pro-metastatic genes such as matrix metalloproteinases and through the stimulation of adjacent cells to produce angiogenic proteins and growth factors such as VEGF, IL-8, IL-6, TNFalpha, and TGFbeta. The IL-1 receptor antagonist (IL-1ra) is a naturally occurring inhibitor to IL-1 and acts by binding to the IL-1 receptor without activating it. The protein has been shown to decrease tumor growth, angiogenesis, and metastases in murine xenograft models. Our focus in this review is to summarize the known data on the role of IL-1 in tumor progression and metastasis and the use of IL-1 inhibition as a novel therapeutic approach in the treatment of solid organ malignancies.

Multipotent cancer stem cells derived from human malignant peritoneal mesothelioma promote tumorigenesis.

During the progression of malignant peritoneal mesothelioma (MPeM), tumor nodules propagate diffusely within the abdomen and tumors are characterized by distinct phenotypic sub-types. Recent studies in solid organ cancers have shown that cancer stem cells (CSCs) play a pivotal role in the initiation and progression of tumors. However, it is not known whether tumorigenic stem cells exist and whether they promote tumor growth in MPeM. In this study, we developed and characterized a CSC model for MPeM using stably expandable tumorigenic stem cells derived from patient tumors. We found morphologically distinct populations of CSCs that divide asymmetrically or symmetrically in MPeM in vitro cell culture. The MPeM stem cells (MPeMSCs) express stem cell markers c-MYC, NES and VEGFR2 and in the presence of matrix components cells form colony spheres. MPeMSCs are multipotent, differentiate into neuronal, vascular and adipose progeny upon defined induction and the differentiating cells express lineage-specific markers such as TUBB3, an early neuronal marker; vWF, VEGFA, VEGFC and IL-8, endothelial markers; and PPARγ and FABP4, adipose markers. Xenotransplantation experiments using MPeMSCs demonstrated early tumor growth compared with parental cells. Limiting dilution experiments using MPeMSCs and endothelial lineage-induced cells derived from a single MPeMSC resulted in early tumor growth in the latter group indicating that endothelial differentiation of MPeMSCs is important for MPeM tumor initiation. Our observation that the MPeM tumors contain stem cells with tumorigenic potential has important implications for understanding the cells of origin and tumor progression in MPeM and hence targeting CSCs may be a useful strategy to inhibit malignant progression.

Activation of the phosphoinositide-3-kinase and mammalian target of rapamycin signaling pathways are associated with shortened survival in patients with malignant peritoneal mesothelioma.

BACKGROUND: Malignant peritoneal mesothelioma (MPM) is a rare malignancy of the serosal membranes of the abdominal cavity. This cancer is ultimately fatal in almost all afflicted individuals; however, there is marked variability in its clinical behavior: Some patients die rapidly, and others survive for many years. In the current study, the authors investigated the molecular nature of MPM to obtain insights into the heterogeneity of its clinical behavior and to identify new therapeutic targets for intervention. METHODS: Fresh pretreatment tumor samples were collected from 41 patients with MPM who underwent surgical cytoreduction and received regional intraoperative chemotherapy perfusion. From those samples, gene expression analyses were performed. The major cellular pathways that were identified in this cancer were inhibited using a pathway-specific inhibitor. RESULTS: Unsupervised clustering of genes identified 2 distinct groups of patients with significantly different survivals (Group A: median survival, 24 months; Group B: median survival, 69.5 months; P = .035). Phosphoinositide-3-kinase (PI3K) and the closely interacting mammalian target of rapamycin (mTOR) signaling pathways were overexpressed predominantly in the poor survival group; and the genes of these pathways, phosphoinositide-3-kinase, catalytic, α polypeptide (PIK3CA) and rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR), were highly significantly predictive of shortened patient survival in Group A. The role of these pathways in MPM tumor progression was also investigated by treating 2 MPM cell lines with BEZ235, a dual-class PI3K and mTOR inhibitor, and the authors observed significant inhibition of downstream cell signaling and cell proliferation. CONCLUSIONS: Taken together, the results from this study revealed that, based on gene expression profiles, there were 2 distinct patient groups with significantly different survival and that targeting the PI3K and mTOR signaling pathways may have significant therapeutic value in patients with MPM.