Discovery of T-1101 tosylate as a first-in-class clinical candidate for Hec1/Nek2 inhibition in cancer therapy.

Highly expressed in cancer 1 (Hec1) plays an essential role in mitosis and is correlated with cancer formation, progression, and survival. Phosphorylation of Hec1 by Nek2 kinase is essential for its mitotic function, thus any disruption of Hec1/Nek2 protein-protein interaction has potential for cancer therapy. We have developed T-1101 tosylate (9j tosylate, 9j formerly known as TAI-95), optimized from 4-aryl-N-pyridinylcarbonyl-2-aminothiazole of scaffold 9 by introducing various C-4' substituents to enhance potency and water solubility, as a first-in-class oral clinical candidate for Hec1 inhibition with potential for cancer therapy. T-1101 has good oral absorption, along with potent in vitro antiproliferative activity (IC50: 14.8-21.5 nM). It can achieve high concentrations in Huh-7 and MDA-MB-231 tumor tissues, and showed promise in antitumor activity in mice bearing human tumor xenografts of liver cancer (Huh-7), as well as of breast cancer (BT474, MDA-MB-231, and MCF7) with oral administration. Oral co-administration of T-1101 halved the dose of sorafenib (25 mg/kg to 12.5 mg/kg) required to exhibit comparable in vivo activity towards Huh-7 xenografts. Cellular events resulting from Hec1/Nek2 inhibition with T-1101 treatment include Nek2 degradation, chromosomal misalignment, and apoptotic cell death. A combination of T-1101 with either of doxorubicin, paclitaxel, and topotecan in select cancer cells also resulted in synergistic effects. Inactivity of T-1101 on non-cancerous cells, a panel of kinases, and hERG demonstrates cancer specificity, target specificity, and cardiac safety, respectively. Subsequent salt screening showed that T-1101 tosylate has good oral AUC (62.5 µM·h), bioavailability (F = 77.4%), and thermal stability. T-1101 tosylate is currently in phase I clinical trials as an orally administered drug for cancer therapy.

Finger-powered agglutination lab chip with CMOS image sensing for rapid point-of-care diagnosis applications.

Agglutination is an antigen-antibody reaction with visible expression of aggregation of the antigens and their corresponding antibodies. Applications extend to the identification of acute bacterial infection, hemagglutination, such as blood grouping, and diagnostic immunology. Our finger-powered agglutination lab chip with external CMOS image sensing was developed to support a platform for inexpensive, rapid point-of-care (POC) testing applications related to agglutination effects. In this paper, blood grouping (ABO and Rh grouping) was utilized to demonstrate the function of our finger-powered agglutination lab chip with CMOS image sensing. Blood antibodies were preloaded into the antibody reaction chamber in the lab chip. The blood sample was pushed through the antibody reaction chamber using finger-powered pressure actuation to initiate a hemagglutination reaction to identify the blood type at the on-chip detection area using our homemade CMOS image sensing mini-system. Finger-powered actuation without the need for external electrical pumping is excellent for low-cost POC applications, but the pumping liquid volume per finger push is hard to control. In our finger-powered agglutination lab chip with CMOS image sensing, we minimized the effects of different finger push depths and achieved robust performance for the test results with different push depths. The driving sample volume per finger push is about 0.79 mm3. For different chips and different pushes, the driven sample volume per finger push was observed to vary in the range of 0.64 to 1.18 mm3. The red blood cells were separated from the plasma on-chip after the whole blood sample was finger pumped and before the red blood cells reached the antibody chamber via an embedded plasma-separation membrane. Our homemade CMOS image mini-system robustly read and identified the agglutination results on our agglutination lab chip.

Clinical Evaluation of IntelliPlex™ KRAS G12/13 Mutation Kit for Detection of KRAS Mutations in Codon 12 and 13: A Novel Multiplex Approach.

BACKGROUND: Colorectal cancer (CRC) is among the most frequently occurring cancers worldwide and its incidence is forecasted to increase. Testing for KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations in colorectal tissue biopsy samples has become a crucial tool to guide therapeutic decisions for personalized treatment. OBJECTIVE: The objective of this study was to determine the diagnostic sensitivity and specificity of the IntelliPlex™ KRAS G12/13 Mutation Kit using clinical specimens compared to Sanger sequencing as the reference method. METHODS: A total of 248 formalin-fixed paraffin-embedded (FFPE) tissue samples, with CRC tumors comprising more than 10% of the whole tissue sample, were included in the study and analyzed for specific KRAS mutations in codons 12 and 13. For samples with discordant results between Sanger sequencing and the IntelliPlex™ KRAS G12/13 Mutation Kit, Pyrosequencing was utilized to resolve the KRAS mutational status. RESULTS: Sequencing determined 153 specimens as KRAS wild-type genotype while the IntelliPlex™ KRAS G12/13 Mutation Kit confirmed 139 of the wild-type cases, resulting in a clinical specificity of 90.8% (95% confidence interval (CI) 85.12-94.91). All 95 specimens with a reported mutation in codons 12 or 13 of KRAS by sequencing were also reported as non-wild-type by the IntelliPlex™ KRAS G12/13 Mutation Kit, resulting in a clinical sensitivity to detect KRAS mutations of 100% (95% CI 96.19-100). CONCLUSIONS: The IntelliPlex™ KRAS G12/13 Mutation Kit demonstrates suitable specificity and sensitivity for use in clinical laboratories to determine the mutational status of KRAS codons 12 and 13.

CCN2 promotes drug resistance in osteosarcoma by enhancing ABCG2 expression.

In recent years, osteosarcoma survival rates have failed to improve significantly with conventional treatment modalities because of the development of chemotherapeutic resistance. The human breast cancer resistance protein/ATP binding cassette subfamily G member 2 (BCRP/ABCG2), a member of the ATP-binding cassette family, uses ATP hydrolysis to expel xenobiotics and chemotherapeutics from cells. CCN family member 2 (CCN2) is a secreted protein that modulates the biological function of cancer cells, enhanced ABCG2 protein expression and activation in this study via the α6ß1 integrin receptor and increased osteosarcoma cell viability. CCN2 treatment downregulated miR-519d expression, which promoted ABCG2 expression. In a mouse xenograft model, knockdown of CCN2 expression increased the therapeutic effect of doxorubicin, which was reversed by ABCG2 overexpression. Our data show that CCN2 increases ABCG2 expression and promotes drug resistance through the α6ß1 integrin receptor, whereas CCN2 downregulates miR-519d. CCN2 inhibition may represent a new therapeutic concept in osteosarcoma.

Leukocyte Cell-Derived Chemotaxin 2 Retards Non-Small Cell Lung Cancer Progression Through Antagonizing MET and EGFR Activities.

BACKGROUND/AIMS: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy is a clinical option for non-small cell lung cancer (NSCLC) harboring activating EGFR mutations or for cancer with wild-type (WT) EGFR when chemotherapy has failed. MET receptor activation or MET gene amplification was reported to be a major mechanism of acquired resistance to EGFR-TKI therapy in NSCLC cells. Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that was shown to suppress metastasis of hepatocellular carcinoma via inhibiting MET activity. Until now, the biological function responsible for LECT2's action in human NSCLC remains unclear. METHODS: LECT2-knockout (KO) mice and NOD/SCID/IL2rgnull (NSG) mice were respectively used to investigate the effects of LECT2 on the tumorigenicity and metastasis of murine (Lewis lung carcinoma, LLC) and human (HCC827) lung cancer cells. The effect of LECT2 on in vitro cell proliferation was evaluated, using MTS and colony formation assays. The effect of LECT2 on cell motility was evaluated using transwell migration and invasion assays. An enzyme-linked immunosorbent assay was performed to detect secreted LECT2 in plasma and media. Co-immunoprecipitation and Western blot assays were used to investigate the underlying mechanisms of LECT2 in NSCLC cells. RESULTS: Compared to WT mice, mice with LECT2 deletion exhibited enhanced growth and metastasis of LLC cells, and survival times decreased in LLC-implanted mice. Overexpression of LECT2 in orthotopic human HCC827 xenografts in NSG mice resulted in significant inhibition of tumor growth and metastasis. In vitro, overexpression of LECT2 or treatment with a recombinant LECT2 protein impaired the colony-forming ability and motility of NSCLC cells (HCC827 and PC9) harboring high levels of activated EGFR and MET. Mechanistic investigations found that LECT2 bound to MET and EGFR to antagonize their activation and further suppress their common downstream pathways: phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase. CONCLUSION: EGFR-MET signaling is critical for aggressive behaviors of NSCLC and is recognized as a therapeutic target for NSCLC especially for patients with acquired resistance to EGFR-TKI therapy. Our findings demonstrate, for the first time, that LECT2 functions as a suppressor of the progression of NSCLC by targeting EGFR-MET signaling.

EIF3C-enhanced exosome secretion promotes angiogenesis and tumorigenesis of human hepatocellular carcinoma.

Targeting tumor angiogenesis is a common strategy against human hepatocellular carcinoma (HCC). However, identification of molecular targets as biomarker for elevating therapeutic efficacy is critical to prolong HCC patient survival. Here, we showed that EIF3C (eukaryotic translation initiation factor 3 subunit C) is upregulated during HCC tumor progression and associated with poor patient survival. Expression of EIF3C did not alter proliferation and expression of other tumor progressive genes such as HIF1A, TGFß1 and VEGF, but reduced cell migration in HCC cells. Nevertheless, expression of EIF3C in HCC cells significantly increase secretion of extracellular exosomes confirmed by increased exosomes labelling by PKH26 fluorescent dye, vesicles in exosome size detected by electronic microscopy and nanoparticle tracking analysis, and expression of divergent exosome markers. The EIF3C-increased exosomes were oncogenic to potentiate tumor angiogenesis via tube formation of HUVEC cells and growth of vessels by plugs assays on nude mice. Subcutaneous inoculation of EIF3C-exosomes mixed with Huh7 HCC cells not only promoted growth of vessels but also increased expression of EIF3C in tumors. Conversely, treatment of exosome inhibitor GW4869 reversed aforementioned oncogenic assays. We identified EIF3C activated expression of S100A11 involved in EIF3C-exosome increased tube formation in angiogenesis. Simultaneous high expression of EIF3C and S100A11 in human HCC tumors for RNA level in TCGA and protein level by IHC are associated with poor survival of HCC patients. Collectively, our results demonstrated that EIF3C overexpression is a potential target of angiogenesis for treatment with exosome inhibitor or S100A11 reduction to suppress HCC angiogenesis and tumorigenesis.

Nuclear translocation of PKM2/AMPK complex sustains cancer stem cell populations under glucose restriction stress.

Cancer cells encounter metabolic stresses such as hypoxia and nutrient limitations because they grow and divide more quickly than their normal counterparts. In response to glucose restriction, we found that nuclear translocation of the glycolic enzyme, pyruvate kinase M2 (PKM2), helped cancer cells survive under the metabolic stress. Restriction of glucose stimulated AMPK activation and resulted in co-translocation of AMPK and PKM2 through Ran-mediated nuclear transport. Nuclear PKM2 subsequently bound to Oct4 and promoted the expression of cancer stemness-related genes, which might enrich the cancer stem cell population under the metabolic stress. Nuclear PKM2 was also capable of promoting cancer metastasis in an orthotopic xenograft model. In summary, we found that cytosolic AMPK helped PKM2 carry out its nonmetabolic functions in the nucleus under glucose restriction and that nuclear PKM2 promoted cancer stemness and metastasis. These findings suggested a potential new targeting pathway for cancer therapy in the future.

Expression of 3ß-Hydroxysteroid Dehydrogenase Type 1 in Breast Cancer is Associated with Poor Prognosis Independent of Estrogen Receptor Status.

BACKGROUND: Human 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1) plays a vital role in steroidogenesis in breast tumors and may therefore be a suitable target for treatment of breast cancer. This study investigated the role of HSD3B1 in the pathogenesis of breast cancer in clinical and experimental settings. METHODS: Expression of HSD3B1 in primary tumors of 258 breast cancer patients was evaluated by immunohistochemistry. Screening of breast cancer cell lines indicated that triple-negative MDA-MB-231 cells expressed HSD3B1. The effects from genetic and pharmacologic inhibition of HSD3B1 were assessed in vitro and in vivo. RESULTS: The findings showed that 44% of the 258 breast cancers were HSD3B1-positive. The HSD3B1-positivity was associated with advanced-stage disease (p = 0.009) and reduced recurrence-free survival (p = 0.048) but not with tumor subtype or estrogen receptor status. Silencing of HSD3B1 or treatment with an HSD3B1 inhibitor (trilostane) reduced colony formation in breast cancer cells. Knockdown of HSD3B1 inhibited cell proliferation and migration. Analysis of a murine xenograft tumor model indicated that trilostane significantly slowed tumor growth. CONCLUSIONS: Expression of HSD3B1 in breast cancer is negatively associated with prognosis. The study found HSD3B1 to be a potential therapeutic target for breast cancer independent of estrogen receptor status.

Inhibition of MDA-MB-231 breast cancer cell proliferation and tumor growth by apigenin through induction of G2/M arrest and histone H3 acetylation-mediated p21WAF1/CIP1 expression.

Apigenin (4',5,7-trihydroxyflavone), a flavonoid commonly found in fruits and vegetables, has anticancer properties in various malignant cancer cells. However, the molecular basis of the anticancer effect remains to be elucidated. In this study, we investigated the cellular mechanisms underlying the induction of cell cycle arrest by apigenin. Our results showed that apigenin at the nonapoptotic induction concentration inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in the MDA-MB-231 breast cancer cell line. Immunoblot analysis indicated that apigenin suppressed the expression of cyclin A, cyclin B, and cyclin-dependent kinase-1 (CDK1), which control the G2-to-M phase transition in the cell cycle. In addition, apigenin upregulated p21WAF1/CIP1 and increased the interaction of p21WAF1/CIP1 with proliferating cell nuclear antigen (PCNA), which inhibits cell cycle progression. Furthermore, apigenin significantly inhibited histone deacetylase (HDAC) activity and induced histone H3 acetylation. The subsequent chromatin immunoprecipitation (ChIP) assay indicated that apigenin increased acetylation of histone H3 in the p21WAF1/CIP1 promoter region, resulting in the increase of p21WAF1/CIP1 transcription. In a tumor xenograft model, apigenin effectively delayed tumor growth. In these apigenin-treated tumors, we also observed reductions in the levels of cyclin A and cyclin B and increases in the levels of p21WAF1/CIP1 and acetylated histone H3. These findings demonstrate for the first time that apigenin can be used in breast cancer prevention and treatment through epigenetic regulation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 434-444, 2017.

Multipoint Kras oncogene mutations potentially indicate mucinous carcinoma on the entire spectrum of mucinous ovarian neoplasms.

Kras mutation is a common phenomenon in many human neoplasms. We aimed to assess the Kras mutational status along the histological continuum from normal ovaries to the development of benign, borderline and malignant ovarian mucinous neoplasms. We analyzed 41 cases of malignant, 10 cases of borderline, 7 cases of benign mucinous ovarian tumors and 7 cases of normal ovarian tissue. The prevalence of Kras mutations in the normal ovary was 0.00% (n=0/7), while the prevalence in benign, borderline and malignant mucinous neoplasms was 57.14% (n=4/7), 90.00% (n=9/10) and 75.61% (n=31/41), respectively. Multiple Kras mutations were detected in 6 cases of mucinous carcinoma, including 5 double mutations with G13D/V14I (n=1), G12V/G13S (n=1), G12D/G13S (n=3) and one triple mutation with A11V/G13N/V14I (n=1). We identified six cases with 3 novel Kras mutations not previously described in the COSMIC database, which included A11V (n=3) and V14I (n=2) in mucinous carcinomas, and A11T (n=1) in a mucinous borderline tumor. In conclusion, Kras mutation appears to be one of the imperative events in the ovarian mucinous adenoma-borderline tumor-carcinoma sequence, as increased numbers of Kras mutations have been shown to be the strongest predictor of unequivocal malignancy in ovarian mucinous neoplasms.

Glutaminase 2 stabilizes Dicer to repress Snail and metastasis in hepatocellular carcinoma cells.

Glutaminolysis that catabolizes glutamine to glutamate plays a critical role in cancer progression. Glutaminase 2 (GLS2) has been reported as a tumor suppressor. Recent studies implied that GLS2 may display its multifunction besides classical metabolic feature by different localizations and potential protein binding domains. Here, we showed that GLS2 expression correlates inversely with stage, vascular invasion, tumor size and poor prognosis in human hepatocellular carcinoma (HCC) tissues. We found that GLS2 significantly represses cell migration, invasion and metastasis of HCC through downregulation of Snail in vitro and in vivo. Moreover, our results demonstrated that GLS2 interacts with Dicer and stabilizes Dicer protein to facilitate miR-34a maturation and subsequently represses Snail expression in a glutaminase activity independent manner. Our findings indicate that non-glutaminolysis function of GLS2 inhibits migration and invasion of HCC cells by repressing the epithelial-mesenchymal transition via the Dicer-miR-34a-Snail axis.

Inhibition of VEGF165/VEGFR2-dependent signaling by LECT2 suppresses hepatocellular carcinoma angiogenesis.

Hepatocellular carcinoma (HCC) relies on angiogenesis for growth and metastasis. Leukocyte cell-derived chemotaxin 2 (LECT2) is a cytokine and preferentially expressed in the liver. Previous studies have found that LECT2 targets to both immune and tumor cells to suppress HCC development and vascular invasion. Although LECT2 did not affect HCC cells growth in vitro, it still suppressed HCC xenografts growth in immune-deficient mice, suggesting other cells such as stroma cells may also be targeted by LECT2. Here, we sought to determine the role of LECT2 in tumor angiogenesis in HCC patients. We found that LECT2 expression inhibited tumor growth via angiogenesis in the HCC xenograft model. Specifically, we demonstrated that recombinant human LECT2 protein selectively suppressed vascular endothelial growth factor (VEGF)165-induced endothelial cell proliferation, migration, and tube formation in vitro and in vivo. Mechanistically, LECT2 reduced VEGF receptor 2 tyrosine phosphorylation and its downstream extracellular signal-regulated kinase and AKT phosphorylation. Furthermore, LECT2 gene expression correlated negatively with angiogenesis in HCC patients. Taken together, our findings demonstrate that LECT2 inhibits VEGF165-induced HCC angiogenesis through directly binding to VEGFR2 and has broad applications in treating VEGF-mediated solid tumors.

Regulation of EBV LMP1-triggered EphA4 downregulation in EBV-associated B lymphoma and its impact on patients' survival.

Epstein-Barr virus (EBV), an oncogenic human virus, is associated with several lymphoproliferative disorders, including Burkitt lymphoma, Hodgkin disease, diffuse large B-cell lymphoma (DLBCL), and posttransplant lymphoproliferative disorder (PTLD). In vitro, EBV transforms primary B cells into lymphoblastoid cell lines (LCLs). Recently, several studies have shown that receptor tyrosine kinases (RTKs) play important roles in EBV-associated neoplasia. However, details of the involvement of RTKs in EBV-regulated B-cell neoplasia and malignancies remain largely unclear. Here, we found that erythropoietin-producing hepatocellular receptor A4 (EphA4), which belongs to the largest RTK Eph family, was downregulated in primary B cells post-EBV infection at the transcriptional and translational levels. Overexpression and knockdown experiments confirmed that EBV-encoded latent membrane protein 1 (LMP1) was responsible for this EphA4 suppression. Mechanistically, LMP1 triggered the extracellular signal-regulated kinase (ERK) pathway and promoted Sp1 to suppress EphA4 promoter activity. Functionally, overexpression of EphA4 prevented LCLs from proliferation. Pathologically, the expression of EphA4 was detected in EBV(-) tonsils but not in EBV(+) PTLD. In addition, an inverse correlation of EphA4 expression and EBV presence was verified by immunochemical staining of EBV(+) and EBV(-) DLBCL, suggesting EBV infection was associated with reduced EphA4 expression. Analysis of a public data set showed that lower EphA4 expression was correlated with a poor survival rate of DLBCL patients. Our findings provide a novel mechanism by which EphA4 can be regulated by an oncogenic LMP1 protein and explore its possible function in B cells. The results provide new insights into the role of EphA4 in EBV(+) PTLD and DLBCL.

CCL5 promotes VEGF-C production and induces lymphangiogenesis by suppressing miR-507 in human chondrosarcoma cells.

Chondrosarcoma is the second most frequently occurring type of bone malignancy that is characterized by the distant metastasis propensity. Vascular endothelial growth factor-C (VEGF-C) is the major lymphangiogenic factor, and makes crucial contributions to tumor lymphangiogenesis and lymphatic metastasis. Chemokine CCL5 has been reported to facilitate angiogenesis and metastasis in chondrosarcoma. However, the effect of chemokine CCL5 on VEGF-C regulation and lymphangiogenesis in chondrosarcoma has largely remained a mystery. In this study, we showed a clinical correlation between CCL5 and VEGF-C as well as tumor stage in human chondrosarcoma tissues. We further demonstrated that CCL5 promoted VEGF-C expression and secretion in human chondrosarcoma cells. The conditioned medium (CM) from CCL5-overexpressed cells significantly induced tube formation of human lymphatic endothelial cells (LECs). Mechanistic investigations showed that CCL5 activated VEGF-C-dependent lymphangiogenesis by down-regulating miR-507. Moreover, inhibiting CCL5 dramatically reduced VEGF-C and lymphangiogenesis in the chondrosarcoma xenograft animal model. Collectively, we document for the first time that CCL5 induces tumor lymphangiogenesis by the induction of VEGF-C in human cancer cells. Our present study reveals miR-507/VEGF-C signaling as a novel mechanism in CCL5-mediated tumor lymphangiogenesis. Targeting both CCL5 and VEGF-C pathways might serve as the potential therapeutic strategy to block cancer progression and metastasis in chondrosarcoma.

Castleman disease mimicking nodal recurrence of thyroid cancer.

A 54-year-old woman who had undergone total thyroidectomy and radioactive iodine treatment for papillary thyroid cancer presented with elevated stimulated thyroglobulin levels and negative I-131 scan. Ultrasonography revealed suspicious lateral neck lymph nodes, which were FDG-avid. Neck dissection led to a diagnosis of Castleman disease.

Tricetin suppresses the migration/invasion of human glioblastoma multiforme cells by inhibiting matrix metalloproteinase-2 through modulation of the expression and transcriptional activity of specificity protein 1.

OBJECTIVE: Glioblastoma multiforme (GBM) is a severely invasive tumor that can be fatal because it is difficult to treat. Tricetin, a natural flavonoid, was demonstrated to inhibit the growth of various cancers, but the effect of tricetin on cancer motility is largely unknown. RESEARCH DESIGN AND METHODS: In the present study, we examined the anti-invasive properties of tricetin in huwman GBM cells. RESULTS: Our results showed that tricetin inhibited the migration/invasion of two GBM cell lines. We found that tricetin inhibited MMP-2 expression in the GBM cells. Real-time polymerase chain reaction and promoter activity assays indicated that tricetin inhibited MMP-2 expression at the transcriptional level. Such inhibitory effects were associated with the suppression of specificity protein-1 (SP-1) DNA-binding activity. An examination of clinical samples revealed a positive correlation between SP-1 and MMP-2 in glioma specimens, and higher expression levels were correlated with a worse probability of survival. Moreover, blocking the extracellular signal-regulated kinase (ERK) pathway also inhibited MMP-2-mediated cell motility, and further enhanced the anti-invasive ability of tricetin in GBM cells. CONCLUSIONS: SP-1 is an important target of tricetin for suppressing MMP-2-mediated cell motility in GBM cells, and a combination of tricetin and an ERK inhibitor may be a good strategy for preventing GBM invasion.