Correction: Lin et al. Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma. Cancers 2021, 13, 3367.

There is an omission in the Institutional Review Board Statement and Conflict of Interest statements of the paper [...].

Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma.

Cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of debilitating, incurable malignancies. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes, accounting for ~65% of CTCL cases. Patients with advanced disease have a poor prognosis and low median survival rates of four years. CTCLs develop from malignant skin-homing CD4+ T cells that spread to lymph nodes, blood, bone marrow and viscera in advanced stages. Current treatments options for refractory or advanced CTCL, including chemotherapeutic and biological approaches, rarely lead to durable responses. The exact molecular mechanisms of CTCL pathology remain unclear despite numerous genomic and gene expression profile studies. However, apoptosis resistance is thought to play a major role in the accumulation of malignant T cells. Here we show that NT1721, a synthetic epidithiodiketopiperazine based on a natural product, reduced cell viability at nanomolar concentrations in CTCL cell lines, while largely sparing normal CD4+ cells. Treatment of CTCL cells with NT1721 reduced proliferation and potently induced apoptosis. NT1721 mediated the downregulation of GLI1 transcription factor, which was associated with decreased STAT3 activation and the reduced expression of downstream antiapoptotic proteins (BCL2 and BCL-xL). Importantly, NT1721, which is orally available, reduced tumor growth in two CTCL mouse models significantly better than two clinically used drugs (romidepsin, gemcitabine). Moreover, a combination of NT1721 with gemcitabine reduced the tumor growth significantly better than the single drugs. Taken together, these results suggest that NT1721 may be a promising new agent for the treatment of CTCLs.

Attenuation of hedgehog/GLI signaling by NT1721 extends survival in pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most lethal malignancies due to frequent late diagnosis, aggressive tumor growth and metastasis formation. Continuously raising incidence rates of pancreatic cancer and a lack of significant improvement in survival rates over the past 30 years highlight the need for new therapeutic agents. Thus, new therapeutic agents and strategies are urgently needed to improve the outcome for patients with pancreatic cancer. Here, we evaluated the anti-tumor activity of a new natural product-based epidithiodiketopiperazine, NT1721, against pancreatic cancer. METHODS: We characterized the anticancer efficacy of NT1721 in multiple pancreatic cancer cell lines in vitro and in two orthotopic models. We also compared the effects of NT1721 to clinically used hedgehog inhibitors and the standard-of-care drug, gemcitabine. The effect of NT1721 on hedgehog/GLI signaling was assessed by determining the expression of GLI and GLI target genes both in vitro and in vivo. RESULTS: NT1721 displayed IC50 values in the submicromolar range in multiple pancreatic cancer cell lines, while largely sparing normal pancreatic epithelial cells. NT1721 attenuated hedgehog/GLI signaling through downregulation of GLI1/2 transcription factors and their downstream target genes, which reduced cell proliferation and invasion in vitro and significantly decreased tumor growth and liver metastasis in two preclinical orthotopic mouse models of pancreatic cancer. Importantly, treatment with NT1721 significantly improved survival times of mice with pancreatic cancer compared to the standard-of-care drug, gemcitabine. CONCLUSIONS: Favorable therapeutics properties, i.e. 10-fold lower IC50 values than clinically used hedgehog inhibitors (vismodegib, erismodegib), a 90% reduction in liver metastasis and significantly better survival times compared to the standard-of-care drug, gemcitabine, provide a rational for testing NT1721 in the clinic either as a single agent or possibly in combination with gemcitabine or other therapeutic agents in PDAC patients overexpressing GLI1/2. This could potentially result in promising new treatment options for patients suffering from this devastating disease.

SMC1A is associated with radioresistance in prostate cancer and acts by regulating epithelial-mesenchymal transition and cancer stem-like properties.

Prostate cancer is one of the most commonly diagnosed cancers and a pressing health challenge in men worldwide. Radiation therapy (RT) is widely considered a standard therapy for advanced as well as localized prostate cancer. Although this primary therapy is associated with high cancer control rates, up to one-third of patients undergoing radiation therapy becomes radio-resistant and/or has tumor-relapse/recurrence. Therefore, focus on new molecular targets and pathways is essential to develop novel radio-sensitizing agents for the effective and safe treatment of prostate cancer. Here, we describe functional studies that were performed to investigate the role of structural maintenance of chromosome-1 (SMC1A) in radioresistance of metastatic prostate cancer cells. Short hairpin RNA (shRNA) was used to suppress SMC1A in metastatic castration-resistant prostate cancer cells, DU145 and PC3. Clonogenic survival assays, Western blot, RT-PCR, and γ-H2AX staining were used to assess the effect of SMC1A knockdown on radiation sensitivity of these prostate cancer cells. We demonstrate that SMC1A is overexpressed in human prostate tumors compared to the normal adjacent tissue. SMC1A knockdown limits the clonogenic potential, epithelial-mesenchymal transition (EMT), and cancer stem-like cell (CSC) properties of DU145 and PC3 cells and enhanced efficacy of RT in these cells. Targeted inhibition of SMC1A not only plays a critical role in overcoming radio-resistance in prostate cancer cells, but also suppresses self-renewal and the tumor-propagating potential of x-irradiated cancer cells. We propose that SMC1A could be a potential molecular target for the development of novel radio-sensitizing therapeutic agents for management of radio-resistant metastatic prostate cancer.

Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models.

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N²-1-(carboxyethyl)-2'-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors.

NT1721, a novel epidithiodiketopiperazine, exhibits potent in vitro and in vivo efficacy against acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by heterogeneous genetic and epigenetic changes in hematopoietic progenitors that lead to abnormal self-renewal and proliferation. Despite high initial remission rates, prognosis remains poor for most AML patients, especially for those harboring internal tandem duplication (ITD) mutations in the fms-related tyrosine kinase-3 (FLT3). Here, we report that a novel epidithiodiketopiperazine, NT1721, potently decreased the cell viability of FLT3-ITD+ AML cell lines, displaying IC50 values in the low nanomolar range, while leaving normal CD34+ bone marrow cells largely unaffected. The IC50 values for NT1721 were significantly lower than those for clinically used AML drugs (i.e. cytarabine, sorafenib) in all tested AML cell lines regardless of their FLT3 mutation status. Moreover, combinations of NT1721 with sorafenib or cytarabine showed better antileukemic effects than the single agents in vitro. Combining cytarabine with NT1721 also attenuated the cytarabine-induced FLT3 ligand surge that has been linked to resistance to tyrosine kinase inhibitors. Mechanistically, NT1721 depleted DNA methyltransferase 1 (DNMT1) protein levels, leading to the re-expression of silenced tumor suppressor genes and apoptosis induction. NT1721 concomitantly decreased the expression of EZH2 and BMI1, two genes that are associated with the maintenance of leukemic stem/progenitor cells. In a systemic FLT3-ITD+ AML mouse model, treatment with NT1721 reduced tumor burdens by > 95% compared to the control and significantly increased survival times. Taken together, our results suggest that NT1721 may represent a promising novel agent for the treatment of AML.

TLR9 signaling through NF-κB/RELA and STAT3 promotes tumor-propagating potential of prostate cancer cells.

Prostate cancer progression was associated with tumorigenic signaling activated by proinflammatory mediators. However, the etiology of these events remains elusive. Here, we demonstrate that triggering of the innate immune receptor, Toll-like Receptor 9 (TLR9), in androgen-independent prostate cancer cells initiates signaling cascade leading to increased tumor growth and progression. Using limited dilution/serial transplantation experiments, we show that TLR9 is essential for prostate cancer cells' potential to propagate and self-renew in vivo. Furthermore, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of LNCaP- and PC3-derived prostate cancer cell variants. Genome-wide transcriptional analysis of prostate cancer cells isolated from xenotransplanted TLR9-positive and -negative tumors revealed a unique gene expression signature, with prominent upregulation of inflammation- and stem cell-related markers. TLR9 signaling orchestrated expression of critical stem cell-related genes such as NKX3.1, KLF-4, BMI-1 and COL1A1, at both mRNA and protein levels. Our further analysis identified that TLR9-induced NF-κB/RELA and STAT3 transcription factors co-regulated NKX3.1 and KLF4 gene expression by directly binding to both promoters. Finally, we demonstrated the feasibility of using TLR9-targeted siRNA delivery to block RELA- and STAT3-dependent prostate cancer cell self-renewal in vivo. The intratumoral administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates inhibited growth of established prostate tumors and reduced clonogenic potential of cancer cells. Overcoming cancer cell self-renewal and tumor-propagating potential by targeted inhibition of TLR9 signaling can provide therapeutic strategy for late-stage prostate cancer patients.

Baicalein upregulates DDIT4 expression which mediates mTOR inhibition and growth inhibition in cancer cells.

Baicalein is a natural flavone that exhibits anticancer properties. Using microarrays we found that DDIT4 was the highest transcript induced by baicalein in cancer cells. We confirmed in multiple cancer cell lines large, dose-related expression of DDIT4 by quantitative RT-PCR and immunoblot, which correlates with growth inhibition. Time course experiments demonstrate that DDIT4 is rapidly inducible, with high expression maintained for several days in vitro. Induction of DDIT4 expression is p53 independent based on evaluation of p53 knockout cells. Since DDIT4 is known to inhibit mTORC1 activity we confirmed that baicalein suppresses phosphorylation of mTORC1 targets. Using RNA interference we demonstrate that mTORC1 activity and growth inhibition by baicalein is attenuated by knockdown of DDIT4. We furthermore demonstrate suppression of established tumors by baicalein in a mouse model of breast cancer with increased DDIT4 expression in the tumors. Finally, we demonstrate that baicalein upregulates DDIT4 and causes mTORC1 and growth inhibition in platinum resistant cancer cells in marked contrast to platinum chemotherapy treatment. These studies demonstrate that baicalein inhibits mTORC1 through DDIT4 expression, and may be useful in cancer chemotherapy and chemoprevention.

Targeting JAK1/STAT3 signaling suppresses tumor progression and metastasis in a peritoneal model of human ovarian cancer.

JAK/STAT3 is one of the major signaling pathways that is aberrantly activated in ovarian cancer and associated with tumor progression and poor prognosis in patients with ovarian cancer. In this study, we evaluated the therapeutic potential of targeting JAK/STAT3 signaling in ovarian cancer using a peritoneal dissemination mouse model. We developed this mouse model by injecting a metastatic human ovarian cancer cell line, SKOV3-M-Luc, into the peritoneal cavity of immunodeficient mice. This model displayed a phenotype similar to late-stage ovarian cancer, including extensive peritoneal metastasis and ascites production. The constitutive activation of STAT3 in human ovarian cancer cells appeared to be mediated by an autocrine cytokine loop involving the IL6 family of cytokines and JAK1 kinase. shRNA-mediated knockdown of JAK1 or STAT3 in ovarian cancer cells led to reduced tumor growth, decreased peritoneal dissemination, and diminished ascites production, suggesting a critical role of STAT3 in ovarian cancer progression. Similar results were obtained when a small-molecule inhibitor (JAKi) of the JAK1 kinase was used to treat ovarian cancer in this model. In addition, we found that the expression level of IL6 was correlated with activation of STAT3 in ovarian cancer cells both in vitro and in vivo, suggesting a potential application of IL6 as a biomarker. Altogether, our results demonstrate that targeting JAK1/STAT3, using shRNA knockdown or a small-molecule inhibitor, effectively suppressed ovarian tumor progression and, therefore, could be a potential novel therapeutic approach for treating advanced ovarian cancer.

Human breast cancer metastases to the brain display GABAergic properties in the neural niche.

Dispersion of tumors throughout the body is a neoplastic process responsible for the vast majority of deaths from cancer. Despite disseminating to distant organs as malignant scouts, most tumor cells fail to remain viable after their arrival. The physiologic microenvironment of the brain must become a tumor-favorable microenvironment for successful metastatic colonization by circulating breast cancer cells. Bidirectional interplay of breast cancer cells and native brain cells in metastasis is poorly understood and rarely studied. We had the rare opportunity to investigate uncommonly available specimens of matched fresh breast-to-brain metastases tissue and derived cells from patients undergoing neurosurgical resection. We hypothesized that, to metastasize, breast cancers may escape their normative genetic constraints by accommodating and coinhabiting the neural niche. This acquisition or expression of brain-like properties by breast cancer cells could be a malignant adaptation required for brain colonization. Indeed, we found breast-to-brain metastatic tissue and cells displayed a GABAergic phenotype similar to that of neuronal cells. The GABAA receptor, GABA transporter, GABA transaminase, parvalbumin, and reelin were all highly expressed in breast cancer metastases to the brain. Proliferative advantage was conferred by the ability of breast-to-brain metastases to take up and catabolize GABA into succinate with the resultant formation of NADH as a biosynthetic source through the GABA shunt. The results suggest that breast cancers exhibit neural characteristics when occupying the brain microenvironment and co-opt GABA as an oncometabolite.

TLR9 signaling in the tumor microenvironment initiates cancer recurrence after radiotherapy.

Cancer radiotherapy may be immunogenic, but it is unclear why its immunogenic effects are rarely sufficient to prevent tumor recurrence. Here, we report a novel Toll-like receptor 9 (TLR9)-dependent mechanism that initiates tumor regrowth after local radiotherapy. Systemic inhibition of TLR9, but not TLR4, delayed tumor recurrence in mouse models of B16 melanoma, MB49 bladder cancer, and CT26 colon cancer after localized high-dose tumor irradiation. Soluble factors in the microenvironment of regressing tumors triggered TLR9 signaling in freshly recruited myeloid cells appearing within four days of radiotherapy. The tumorigenic effects of TLR9 depended on MyD88/NF-κB-mediated upregulation of interleukin (IL)-6 expression, which in turn resulted in downstream activation of Jak/STAT3 signaling in myeloid cells. In comparing global gene expression in wild-type, TLR9-, or STAT3-deficient myeloid cells derived from irradiated tumors, we identified a unique set of TLR9/STAT3-regulated genes involved in tumor-promoting inflammation and revascularization. Blocking STAT3 function by two myeloid-specific genetic strategies corrected TLR9-mediated cancer recurrence after radiotherapy. Our results suggest that combining localized tumor irradiation with myeloid cell-specific inhibition of TLR9/STAT3 signaling may help eliminate radioresistant cancers.

Intracellular processing of immunostimulatory CpG-siRNA: Toll-like receptor 9 facilitates siRNA dicing and endosomal escape.

Dicer-substrate siRNAs equipped with CpG oligodeoxyribonucleotides overcome the major hurdle in cell-specific siRNA delivery. The CpG-siRNA molecules are actively internalized by TLR9+ cells, without the need for transfection reagents, leading to RNA interference both in vitro and in vivo. Here, we elucidate the molecular mechanisms of CpG-siRNA processing in target cells. We show that shortly after uptake into early endosomes (EE), CpG and siRNA parts of the conjugate are uncoupled in the presence of Dicer endonuclease. Diced siRNA molecules are translocated from endosomes to endoplasmic reticulum, where they can interact with the RNA interference machinery. We previously observed that even though TLR9 is not involved in CpG-siRNA uptake, it is indispensable for induction of gene silencing. To explain the role of TLR9 in intracellular processing of CpG-siRNA, we used primary macrophages derived from wild-type and Tlr9-deficient mice. Macrophages lacking TLR9 showed extended endosomal colocalization of CpG and siRNA parts of the conjugate. However, Tlr9 ablation did not interfere with the interaction of CpG-siRNA with Dicer as shown by in situ proximity ligation assay. Using CpG-siRNA labeled with pH-sensitive dye, we finally identified that lack of TLR9 in macrophages resulted in significant retention of the siRNA in endosomes. Thus, TLR9 facilitates the critical step following CpG-siRNA uncoupling, which is cytoplasmic release of the diced siRNA. These findings suggest that the class of immunostimulatory siRNAs may benefit from activation of certain endosomal immune receptors, such as TLR9, in augmented gene silencing and therapeutic efficacy.

Co-evolution of breast-to-brain metastasis and neural progenitor cells.

Brain colonization by metastatic tumor cells offers a unique opportunity to investigate microenvironmental influences on the neoplastic process. The bi-directional interplay of breast cancer cells (mesodermal origin) and brain cells (neuroectodermal origin) is poorly understood and rarely investigated. In our patients undergoing neurosurgical resection of breast-to-brain metastases, specimens from the tumor/brain interface exhibited increased active gliosis as previously described. In addition, our histological characterization revealed infiltration of neural progenitor cells (NPCs) both outside and inside the tumor margin, leading us to investigate the cellular and molecular interactions between NPCs and metastases. Since signaling by the TGF-ß superfamily is involved in both developmental neurobiology and breast cancer pathogenesis, we examined the role of these proteins in the context of brain metastases. The brain-metastatic breast cancer cell line MDA-MB-231Br (231Br) expressed BMP-2 at significantly higher levels compared to its matched primary breast cancer cell line MDA-MB-231 (231). Co-culturing was used to examine bi-directional cellular effects and the relevance of BMP-2 overexpression. When co-cultured with NPCs, 231 (primary) tumor cells failed to proliferate over 15 days. However, 231Br (brain metastatic) tumor cells co-cultured with NPCs escaped growth inhibition after day 5 and proliferated, occurring in parallel with NPC differentiation into astrocytes. Using shRNA and gene knock-in, we then demonstrated BMP-2 secreted by 231Br cells mediated NPC differentiation into astrocytes and concomitant tumor cell proliferation in vitro. In xenografts, overexpression of BMP-2 in primary breast cancer cells significantly enhanced their ability to engraft and colonize the brain, thereby creating a metastatic phenotype. Conversely, BMP-2 knockdown in metastatic breast cancer cells significantly diminished engraftment and colonization. The results suggest metastatic tumor cells create a permissive neural niche by steering NPC differentiation toward astrocytes through paracrine BMP-2 signaling.

TLR9-mediated siRNA delivery for targeting of normal and malignant human hematopoietic cells in vivo.

STAT3 operates in both cancer cells and tumor-associated immune cells to promote cancer progression. As a transcription factor, it is a highly desirable but difficult target for pharmacologic inhibition. We have recently shown that the TLR9 agonists CpG oligonucleotides can be used for targeted siRNA delivery to mouse immune cells. In the present study, we demonstrate that a similar strategy allows for targeted gene silencing in both normal and malignant human TLR9(+) hematopoietic cells in vivo. We have developed new human cell-specific CpG(A)-STAT3 siRNA conjugates capable of inducing TLR9-dependent gene silencing and activation of primary immune cells such as myeloid dendritic cells, plasmacytoid dendritic cells, and B cells in vitro. TLR9 is also expressed by several human hematologic malignancies, including B-cell lymphoma, multiple myeloma, and acute myeloid leukemia. We further demonstrate that oncogenic proteins such as STAT3 or BCL-X(L) are effectively knocked down by specific CpG(A)-siRNAs in TLR9(+) hematologic tumor cells in vivo. Targeting survival signaling using CpG(A)-siRNAs inhibits the growth of several xenotransplanted multiple myeloma and acute myeloid leukemia tumors. CpG(A)-STAT3 siRNA is immunostimulatory and nontoxic for normal human leukocytes in vitro. The results of the present study show the potential of using tumoricidal/immunostimulatory CpG-siRNA oligonucleotides as a novel 2-pronged therapeutic strategy for hematologic malignancies.

Identification of a natural compound by cell-based screening that enhances interferon regulatory factor-1 activity and causes tumor suppression.

The transcription factor interferon regulatory factor-1 (IRF-1) is induced by many tumor-suppressive stimuli and can mediate antiproliferative and proapoptotic effects in cancer cells. Thus, identifying agents that enhance IRF-1 activity may be an effective approach to cancer therapy. A cell-based screening assay was developed to identify extracts and compounds that could enhance IRF-1 activity, using an IRF-1-dependent luciferase reporter cell line. Through this approach, we identified a natural product extract and a known active component of this extract, baicalein, which causes a marked increase in IRF-1-dependent reporter gene expression and IRF-1 protein, with modulation of known IRF-1 targets PUMA and cyclin D1. Baicalein causes suppression of growth in vitro in multiple cancer cell lines in the low micromolar range. IRF-1 plays a role in this growth suppression as shown by significant resistance to growth suppression in a breast cancer cell line stably transfected with short hairpin RNA against IRF-1. Finally, intraperitoneal administration of baicalein by repeated injection causes inhibition of growth in both xenogeneic and syngeneic mouse models of cancer without toxicity to the animals. These findings indicate that identifying enhancers of IRF-1 activity may have utility in anticancer therapies and that cell-based screening for activation of transcription factors can be a useful approach for drug discovery.

Humanized Lewis-Y specific antibody based delivery of STAT3 siRNA.

The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Le(y)), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Le(y)-expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Le(y)-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a noncovalent construct based on (d-arginine)(9) (9r) modified hu3S193. Le(y)-specific binding and internalization of both the covalent and noncovalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the noncovalent system led to efficient STAT3 silencing in Le(y)-positive cancer cells (A431) but not in Le(y)-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified noncovalent construct induced ∼70% STAT3 knockdown at submicromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ∼50% inhibition of cell proliferation of Le(y)-positive cells. Noncovalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.

Differential expression patterns of capping protein, protein phosphatase 1, and casein kinase 1 may serve as diagnostic markers for malignant melanoma.

Early and accurate diagnosis of malignant melanoma is critical for patient survival. However, currently used diagnostic markers are insufficiently specific, which limits their utility. We aimed to identify molecular markers that are more specific to malignant melanoma, thereby aiding in melanoma diagnosis and treatment. A PCR-based suppression subtractive hybridization was used to identify capping protein Z-line α1, protein phosphatase 1 catalytic subunit ß isoform (PP1CB), and casein kinase 1 α1 (CSNK1A1) as being differentially expressed between melanoma cells and normal melanocytes. Quantitative reverse transcription-PCR and western blot analysis confirmed that these genes were overexpressed in melanoma cells. In addition, immunohistochemical assays revealed that the expression of PP1CB and CSNK1A1 was significantly greater in human melanoma specimens than nevi (P<0.0001). Combined application of PP1CB and CSNK1A showed high sensitivity and specificity for melanoma. Thus, our data suggest that PP1CB and CSNK1A1 are potential biomarkers for distinguishing malignant melanoma from other melanocytic lesions. In addition, because capping protein Z-line α1, PP1CB, and CSNK1A1 are involved in cell motility, which underlies invasion and metastasis of human cancer; they may be novel targets for antimetastatic therapies as well.

Combining histone deacetylase inhibitor vorinostat with aurora kinase inhibitors enhances lymphoma cell killing with repression of c-Myc, hTERT, and microRNA levels.

MK-0457 and MK-5108 are novel aurora kinase inhibitors (AKi) leading to G(2)-M cell-cycle arrest. Growth and survival of multiple lymphoma cell lines were studied with either drug alone or in combination with vorinostat, a histone deacetylase inhibitor (HDACi), using MTS and Annexin V assays, followed by molecular studies. Either of the AKi alone at 100 to 500 nmol/L resulted in approximately 50% reduced cell growth and 10% to 40% apoptosis. Addition of vorinostat reactivated proapoptotic genes and enhanced lymphoma cell death. Quantitative PCR and immunoblotting revealed that epigenetic and protein acetylation mechanisms were responsible for this activity. The prosurvival genes Bcl-X(L) and hTERT were downregulated 5-fold by combination drug treatment, whereas the proapoptotic BAD and BID genes were upregulated 3-fold. The p53 tumor suppressor was stabilized by an increased acetylation in response to vorinostat and a reduced Ser315 phosphorylation in response to aurora kinase A. Vorinostat or trichostatin A decreased MYC mRNA and protein as well as c-Myc-regulated microRNAs. MYC is a critical gene in these responses, as MYC knockdown combined with the expression of the c-Myc antagonist MXD1 raised cell sensitivity to the effects of either AKi. Thus, the HDACi vorinostat leads to both transcriptional and posttranscriptional changes to create a proapoptotic milieu, sensitizing cells to mitosis-specific agents such as AKis.

The JAK2 inhibitor AZD1480 potently blocks Stat3 signaling and oncogenesis in solid tumors.

Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.

Sorafenib inhibits signal transducer and activator of transcription 3 signaling associated with growth arrest and apoptosis of medulloblastomas.

Medulloblastomas are the most frequent malignant brain tumors in children. Sorafenib (Nexavar, BAY43-9006), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in a variety of tumor cells. Sorafenib inhibited proliferation and induced apoptosis in two established cell lines (Daoy and D283) and a primary culture (VC312) of human medulloblastomas. In addition, sorafenib inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) in both cell lines and primary tumor cells. The inhibition of phosphorylated STAT3 (Tyr(705)) occurs in a dose- and time-dependent manner. In contrast, AKT (protein kinase B) was only decreased in D283 and VC312 medulloblastoma cells and mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2) were not inhibited by sorafenib in these cells. Both D-type cyclins (D1, D2, and D3) and E-type cyclin were down-regulated by sorafenib. Also, expression of the antiapoptotic protein Mcl-1, a member of the Bcl-2 family, was decreased and correlated with apoptosis induced by sorafenib. Finally, sorafenib suppressed the growth of human medulloblastoma cells in a mouse xenograft model. Together, our data show that sorafenib blocks STAT3 signaling as well as expression of cell cycle and apoptosis regulatory proteins, associated with inhibition of cell proliferation and induction of apoptosis in medulloblastomas. These findings provide a rationale for treatment of pediatric medulloblastomas with sorafenib.