Dichloroacetate and Quercetin Prevent Cell Proliferation, Induce Cell Death and Slow Tumor Growth in a Mouse Model of HPV-Positive Head and Neck Cancer.

Elevated glucose uptake and production of lactate are common features of cancer cells. Among many tumor-promoting effects, lactate inhibits immune responses and is positively correlated with radioresistance. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase that decreases lactate production. Quercetin is a flavonoid compound found in fruits and vegetables that inhibits glucose uptake and lactate export. We investigated the potential role and mechanisms of DCA, quercetin, and their combination, in the treatment of HPV-positive head and neck squamous cell carcinoma, an antigenic cancer subtype in need of efficacious adjuvant therapies. C57Bl/6-derived mouse oropharyngeal epithelial cells, a previously developed mouse model that was retrovirally transduced with HPV type-16 E6/E7 and activated Ras, were used to assess these compounds. Both DCA and quercetin inhibited colony formation and reduced cell viability, which were associated with mTOR inhibition and increased apoptosis through enhanced ROS production. DCA and quercetin reduced tumor growth and enhanced survival in immune-competent mice, correlating with decreased proliferation as well as decreased acidification of the tumor microenvironment and reduction of Foxp (+) Treg lymphocytes. Collectively, these data support the possible clinical application of DCA and quercetin as adjuvant therapies for head and neck cancer patients.

Ability of metformin to deplete NAD+ contributes to cancer cell susceptibility to metformin cytotoxicity and is dependent on NAMPT expression.

Background: Nicotinamide adenine dinucleotide (NAD+) is vital for not only energy metabolism but also signaling pathways. A major source of NAD+ depletion is the activation of poly (ADP-ribose) polymerase (PARP) in response to DNA damage. We have previously demonstrated that metformin can cause both caspase-dependent cell death and PARP-dependent cell death in the MCF7 breast cancer cells but not in the MDA-MB-231 (231) breast cancer cells while in high-glucose media. We hypothesize that depletion of NAD+ in MCF7 cells via activation of PARP contributes to the cell death caused by metformin. Nicotinamide phosphoribosyltransferase (NAMPT), a key rate-limiting step in converting nicotinamide (vitamin B3) into NAD+, is essential for regenerating NAD+ for normal cellular processes. Evidence shows that overexpression of NAMPT is associated with tumorigenesis. We hypothesize that NAMPT expression may determine the extent to which cancer cells are sensitive to metformin. Results: In this study, we found that metformin significantly decreases NAD+ levels over time, and that this could be delayed by PARP inhibitors. Pretreatment with NAD+ in MCF7 cells also prevents cell death and the enlargement of mitochondria and protects mitochondria from losing membrane potential caused by metformin. This leads to MCF7 cell resistance to metformin cytotoxicity in a manner similar to 231 cells. By studying the differences in NAD+ regulation in these two breast cancer cell lines, we demonstrate that NAMPT is expressed at higher levels in 231 cells than in MCF7 cells. When NAMPT is genetically repressed in 231 cells, they become much more sensitive to metformin-induced cell death. Conversely, overexpressing NAMPT in HEK-293 (293) cells causes the cells to be more resistant to metformin's growth inhibitory effects. The addition of a NAMPT activator also decreased the sensitivity of MCF7 cells to metformin, while the NAMPT activator, P7C3, protects against metformin-induced cytotoxicity. Conclusions: Depletion of cellular NAD+ is a key aspect of sensitivity of cancer cells to the cytotoxic effects of metformin. NAMPT plays a key role in maintaining sufficient levels of NAD+, and cells that express elevated levels of NAMPT are resistant to killing by metformin.

Inhibition of metastatic brain cancer in Sonic Hedgehog medulloblastoma using caged nitric oxide albumin nanoparticles.

Medulloblastoma is a tumor of the cerebellum that metastasizes to the leptomeninges of the central nervous system (CNS), including to forebrain and to spinal cord. The inhibitory effect of polynitroxylated albumin (PNA), a caged nitroxide nanoparticle, on leptomeningeal dissemination and metastatic tumor growth was studied in a Sonic Hedgehog transgenic mouse model. PNA treated mice showed an increased lifespan with a mean survival of 95 days (n = 6, P<0.05) compared with 71 days in controls. In primary tumors, proliferation was significantly reduced and differentiation was significantly increased (P<0.001) as shown by Ki-67+ and NeuN+ immunohistochemistry, while cells in spinal cord tumors appeared unaffected. Yet, histochemical analysis of metastatic tumor in spinal cord showed that the mean total number of cells in spinal cord was significantly reduced in mice treated with PNA compared to albumin vehicle (P<0.05). Examination of various levels of the spinal cord showed that PNA treated mice had significantly reduced metastatic cell density in the thoracic, lumbar and sacral spinal cord levels (P<0.05), while cell density in the cervical region was not significantly changed. The mechanism by which PNA may exert these effects on CNS tumors is discussed.

BASIN: A Semi-automatic Workflow, with Machine Learning Segmentation, for Objective Statistical Analysis of Biomedical and Biofilm Image Datasets.

Micrograph comparison remains useful in bioscience. This technology provides researchers with a quick snapshot of experimental conditions. But sometimes a two- condition comparison relies on researchers' eyes to draw conclusions. Our Bioimage Analysis, Statistic, and Comparison (BASIN) software provides an objective and reproducible comparison leveraging inferential statistics to bridge image data with other modalities. Users have access to machine learning-based object segmentation. BASIN provides several data points such as images' object counts, intensities, and areas. Hypothesis testing may also be performed. To improve BASIN's accessibility, we implemented it using R Shiny and provided both an online and offline version. We used BASIN to process 498 image pairs involving five bioscience topics. Our framework supported either direct claims or extrapolations 57% of the time. Analysis results were manually curated to determine BASIN's accuracy which was shown to be 78%. Additionally, each BASIN version's initial release shows an average 82% FAIR compliance score.

Reduction of Metastasis via Epigenetic Modulation in a Murine Model of Metastatic Triple Negative Breast Cancer (TNBC).

This study investigates the effects of a dual selective Class I histone deacetylase (HDAC)/lysine-specific histone demethylase 1A (LSD1) inhibitor known as 4SC-202 (Domatinostat) on tumor growth and metastasis in a highly metastatic murine model of Triple Negative Breast Cancer (TNBC). 4SC-202 is cytotoxic and cytostatic to the TNBC murine cell line 4T1 and the human TNBC cell line MDA-MB-231; the drug does not kill the normal breast epithelial cell line MCF10A. Furthermore, 4SC-202 reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model, which closely mimics human TNBC in terms of sites of metastasis, reveal reduced tumor burden and lung metastasis. The mechanism of action of 4SC-202 may involve effects on cancer stem cells (CSC) which can self-renew and form metastatic lesions. Approximately 5% of the total 4T1 cell population grown in three-dimensional scaffolds had a distinct CD44high/CD24low CSC profile which decreased after treatment. Bulk transcriptome (RNA) sequencing analyses of 4T1 tumors reveal changes in metastasis-related pathways in 4SC-202-treated tumors, including changes to expression levels of genes implicated in cell migration and cell motility. In summary, 4SC-202 treatment of tumors from a highly metastatic murine model of TNBC reduces metastasis and warrants further preclinical studies.

Lactate induces PD-L1 in HRASG12V-positive oropharyngeal squamous cell carcinoma.

Intratumoral lactate production negatively correlates with survival and tumor clearance in the setting of human papillomavirus positive oropharyngeal squamous cell carcinoma (HPV-positive OPSCC). Robust anti-tumor immune activity is required for tumor clearance in human patients and animal models of this disease, and intratumoral lactate interferes with this process. While lactate is known to directly inhibit T cell activity, recent evidence has demonstrated that lactate can affect gene expression in multiple cell types. We therefore sought to determine if lactate in the tumor microenvironment could aid immune evasion by inducing the expression of immune checkpoint co-inhibitors. Using a mouse cell line transformed with HPV16 E6, E7, and HRASG12V, we determined that OPSCC cells carrying the HRASG12V mutant showed significantly increased expression of PD-L1 in the presence of extracellular lactate. Furthermore, we demonstrate here that lactate activates the MEK/ERK pathway in Ras-mutated cells.

Analysis of Dual Class I Histone Deacetylase and Lysine Demethylase Inhibitor Domatinostat (4SC-202) on Growth and Cellular and Genomic Landscape of Atypical Teratoid/Rhabdoid.

Central nervous system atypical teratoid/rhabdoid tumors (ATRTs) are rare and aggressive tumors with a very poor prognosis. Current treatments for ATRT include resection of the tumor, followed by systemic chemotherapy and radiation therapy, which have toxic side effects for young children. Gene expression analyses of human ATRTs and normal brain samples indicate that ATRTs have aberrant expression of epigenetic markers including class I histone deacetylases (HDAC's) and lysine demethylase (LSD1). Here, we investigate the effect of a small molecule epigenetic modulator known as Domatinostat (4SC-202), which inhibits both class I HDAC's and Lysine Demethylase (LSD1), on ATRT cell survival and single cell heterogeneity. Our findings suggest that 4SC-202 is both cytotoxic and cytostatic to ATRT in 2D and 3D scaffold cell culture models and may target cancer stem cells. Single-cell RNA sequencing data from ATRT-06 spheroids treated with 4SC-202 have a reduced population of cells overexpressing stem cell-related genes, including SOX2. Flow cytometry and immunofluorescence on 3D ATRT-06 scaffold models support these results suggesting that 4SC-202 reduces expression of cancer stem cell markers SOX2, CD133, and FOXM1. Drug-induced changes to the systems biology landscape are also explored by multi-omics enrichment analyses. In summary, our data indicate that 4SC-202 has both cytotoxic and cytostatic effects on ATRT, targets specific cell sub-populations, including those with cancer stem-like features, and is an important potential cancer therapeutic to be investigated in vivo.

Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer.

Of the deaths attributed to cancer, 90% are due to metastasis. Treatments that prevent or cure metastasis remain elusive. Low expression of extracellular superoxide dismutase (EcSOD or SOD3) has been associated with poor outcomes and increased metastatic potential in multiple types of cancer. Here, we characterize the antimetastatic therapeutic mechanisms of a macromolecular extracellular SOD3-mimetic polynitroxyl albumin (PNA, also known as VACNO). PNA is macromolecular human serum albumin conjugated with multiple nitroxide groups and acts as an SOD-mimetic. Here we show that PNA works as a SOD3-mimetic in a highly metastatic 4T1 mouse model of triple negative breast cancer (TNBC). In vitro, PNA dose dependently inhibited 4T1 proliferation, colony formation, and reactive oxygen species (ROS) formation. In vivo, PNA enhanced reperfusion time in the hypoxic cores of 4T1 tumors as measured by ultrasound imaging. Furthermore, PNA enhanced ultrasound signal intensity within the cores of the 4T1 tumors, indicating PNA can increase blood flow and blood volume within the hypoxic cores of tumors. Lung metastasis from 4T1 flank tumor was inhibited by PNA in the presence or absence of doxorubicin, a chemotherapy agent that produces superoxide and promotes metastasis. In a separate study, PNA increased the survival of mice with 4T1 flank tumors when used in conjunction with three standard chemotherapy drugs (paclitaxel, doxorubicin, and cyclophosphamide), as compared to treatment with chemotherapy alone. In this study, PNA-increased survival was also correlated with reduction of lung metastasis. These results support the hypothesis that PNA works through the inhibition of extracellular superoxide/ROS production leading to the conversion of 4T1 cells from a metastatic tumorigenic state to a cytostatic state. These findings support future clinical trials of PNA as an antimetastatic SOD3-mimetic drug to increase overall survival in TNBC patients.

4SC-202 as a Potential Treatment for the Pediatric Brain Tumor Medulloblastoma.

This project involves an examination of the effect of the small molecule inhibitor 4SC-202 on the growth of the pediatric brain cancer medulloblastoma. The small molecule inhibitor 4SC-202 significantly inhibits the viability of the pediatric desmoplastic cerebellar human medulloblastoma cell line DAOY, with an IC50 = 58.1 nM, but does not affect the viability of noncancerous neural stem cells (NSC). 4SC-202 exposure inhibits hedgehog expression in the DAOY cell line. Furthermore, microarray analysis of human medulloblastoma patient tumors indicate significant upregulation of key targets in the Hedgehog signaling pathway and Protein Tyrosine Kinase (PTK7).

Therapeutic Targeting of PTK7 is Cytotoxic in Atypical Teratoid Rhabdoid Tumors.

Novel discoveries involving the evaluation of potential therapeutics are based on newly identified molecular targets for atypical teratoid rhabdoid tumors (ATRT), which are the most common form of infantile brain tumors. Central nervous system ATRTs are rare, aggressive, and fast growing tumors of the brain and spinal cord and carry a very poor prognosis. Currently, the standard of care for ATRT patients is based on surgical resection followed by systemic chemotherapy and radiotherapy, which result in severe side effects. As protein tyrosine kinases have proven to be actionable targets that reduce tumor growth in a number of cancers, we examined how inhibiting tyrosine kinases affected ATRT tumor growth. Here, we examine the therapeutic efficacy of the broad-spectrum tyrosine kinase inhibitor vatalanib in the treatment of ATRT. Vatalanib significantly reduced the growth of ATRT tumor cell lines, both in two-dimensional cell culture and in three-dimensional cell culture using a spheroid model. As vatalanib had a remarkable effect on the growth of ATRT, we decided to use a transcriptomic approach to therapy by examining new actionable targets, such as tyrosine kinases. Next-generation RNA-sequencing and NanoString data analysis showed a significant increase in PTK7 RNA expression levels in ATRT tumors. Inhibition of PTK7 by siRNA treatment significantly decreases the viability of ATRT patient-derived tumor cell lines.Implications: These studies provide the groundwork for future preclinical in vivo studies aiming to investigate the efficacy of PTK7 inhibition on ATRT tumor growth. Mol Cancer Res; 15(8); 973-83. ©2017 AACR.

Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors.

The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition.

Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.

Anti-apoptotic Bcl2 family proteins such as Bcl-x(L) protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-x(L) enhances the efficiency of energy metabolism. Our evidence indicates that Bcl-x(L)interacts directly with the ß-subunit of the F(1)F(O) ATP synthase, decreasing an ion leak within the F(1)F(O) ATPase complex and thereby increasing net transport of H(+) by F(1)F(O) during F(1)F(O) ATPase activity. By patch clamping submitochondrial vesicles enriched in F(1)F(O) ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-x(L) activity increases the membrane leak conductance. In addition, recombinant Bcl-x(L) protein directly increases the level of ATPase activity of purified synthase complexes, and inhibition of endogenous Bcl-x(L) decreases the level of F(1)F(O) enzymatic activity. Our findings indicate that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-x(L)-expressing neurons.

Modulation of a Schistosoma mansoni multidrug transporter by the antischistosomal drug praziquantel.

P-glycoprotein (Pgp) is an ATP-dependent efflux pump involved in transport of xenobiotics from cells that, when overexpressed, can mediate multidrug resistance in mammalian cells. Pgp may be a candidate target for new anthelmintics, as it plays critical roles in normal cell physiology, in removal of drugs from cells, and potentially in the development of drug resistance. Schistosomes are parasitic flatworms that cause schistosomiasis, which affects hundreds of millions of people worldwide. Here, we express SMDR2, a Pgp homologue from Schistosoma mansoni (Platyhelminthes), in Chinese hamster ovary (CHO) cells and use fluorescence-based assays to examine the functional and pharmacological properties of this transporter. Membrane vesicles from stably transfected CHO cells expressing recombinant SMDR2 show significant increases in rhodamine transport and ATP hydrolysis compared with those from control cells or cells transfected with empty vector. SMDR2-mediated transport is inhibited by the Pgp modulators verapamil (IC(50)=12.1 muM) and nifedipine, and also by praziquantel, the current drug of choice against schisotosomiasis (IC(50)=17.4 muM). Efflux measurements of a fluorescent analog of praziquantel indicate that it is also a substrate for SMDR2. The interaction of praziquantel with SMDR2 may offer new strategies for potentiating the action of praziquantel and possibly overcoming drug resistance.

Schistosoma mansoni P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility.

One potential physiological target for new antischistosomals is the parasite's system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding-cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, increased expression of Pgp is associated with multidrug resistance in tumor cells. Pgp may also play a role in drug resistance in helminths. In this report, we examine the relationship between praziquantel (PZQ), the current drug of choice against schistosomiasis, and Pgp expression in Schistosoma mansoni. We show that levels of RNA for SMDR2, a Pgp homolog from S. mansoni, increase transiently in adult male worms following exposure to sub-lethal concentrations (100-500 nM) of PZQ. A corresponding, though delayed, increase in anti-Pgp immunoreactive protein expression occurs in adult males following exposure to PZQ. The level of anti-Pgp immunoreactivity in particular regions of adult worms also increases in response to PZQ. Adult worms from an Egyptian S. mansoni isolate with reduced sensitivity to PZQ express increased levels of SMDR2 RNA and anti-Pgp-immunoreactive protein, perhaps indicating a role for multidrug resistance proteins in development or maintenance of PZQ resistance.

Artepillin C (ARC) in Brazilian green propolis selectively blocks oncogenic PAK1 signaling and suppresses the growth of NF tumors in mice.

There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.

Treatment of schwannomas with an oncolytic recombinant herpes simplex virus in murine models of neurofibromatosis type 2.

Gene therapy for schwannomas was evaluated in two mouse models of neurofibromatosis type 2 (NF2): (1) a transgenic model in which mice express a dominant mutant form of merlin and spontaneously develop schwannomas, and (2) a xenograft model in which human schwannoma tissue is implanted subcutaneously into immune- compromised mice. In both models, schwannoma volumes were monitored by magnetic resonance imaging (MRI) and showed strong gadolinium enhancement typical of these tumors in humans. Both types of tumor were positive for the Schwann cell marker S100, and highly infectable with herpes simplex virus (HSV) vectors. Schwannomas were injected with an oncolytic HSV-1 recombinant virus vector, G47Delta, which has deletions in genes for ribonucleotide reductase (ICP6), gamma34.5, and ICP47. In the NF2 transgenic model, schwannomas were reduced by more than half their original size by 10 days after infection. In the case of subcutaneous schwannoma xenografts, reduction in size after infection occurred more slowly, with a mean reduction of onethird by 42 days after treatment. Schwannomas injected with control vehicles continued to grow slowly over time in both schwannoma models. These studies demonstrate the ability of an oncolytic recombinant HSV vector to reduce the volume of schwannoma tumors in NF2 tumor models in mice and extend the possible therapeutic applications of oncolytic vectors for benign tumors to reduce mass while minimizing nerve damage.

A novel method for imaging apoptosis using a caspase-1 near-infrared fluorescent probe.

Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF) probe selective for caspase-1 (interleukin 1beta-converting enzyme, ICE). This biocompatible, optically quenched ICE-NIRF probe incorporates a peptide substrate, which can be selectively cleaved by caspase-1, resulting in the release of fluorescence signal. The specificity of this probe for caspase-1 is supported by various lines of evidence: 1) activation by purified caspase-1, but not another caspase in vitro; 2) activation of the probe by infection of cells with a herpes simplex virus amplicon vector (HGC-ICE-lacZ) expressing a catalytically active caspase-1-lacZ fusion protein; 3) inhibition of HGC-ICE-lacZ vector-induced activation of the probe by coincubation with the caspase-1 inhibitor YVAD-cmk, but not with a caspase-3 inhibitor; and 4) activation of the probe following standard methods of inducing apoptosis with staurosporine, ganciclovir, or ionizing radiation in culture. These results indicate that this novel ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells. Furthermore, tumor implant experiments indicate that this ICE-NIRF probe can be used to detect caspase-1 activity in living animals. This novel ICE-NIRF probe should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo.

Comparative pathology of nerve sheath tumors in mouse models and humans.

Despite the progress made in our understanding of the biology of neurofibromatosis (NF), the long-term clinical outcome for affected patients has not changed significantly in the past decades, and both NF1 and NF2 are still associated with a significant morbidity and a decreased life span. A number of NF1 and NF2 murine models have been generated to aid in the study of NF tumor biology and in the development of targeted therapies for NF patients. A single, universal pathological classification of the lesions generated in these murine models is essential for the validation of the models, for their analysis and comparison with other models, and for their future effective use in preclinical treatment trials. For the formulation of a pathological classification of these lesions, the WHO classification of human tumors was used as a reference. However, it was not adopted for the classification of the GEM lesions because of some important differences between the human and murine lesions. A novel classification scheme for peripheral nerve sheath tumors in murine models was therefore devised.