A novel chemo-phenotypic method identifies mixtures of salpn, vitamin D3, and pesticides involved in the development of colorectal and pancreatic cancer.

Environmental chemical (EC) exposures and our interactions with them has significantly increased in the recent decades. Toxicity associated biological characterization of these chemicals is challenging and inefficient, even with available high-throughput technologies. In this report, we describe a novel computational method for characterizing toxicity, associated biological perturbations and disease outcome, called the Chemo-Phenotypic Based Toxicity Measurement (CPTM). CPTM is used to quantify the EC "toxicity score" (Zts), which serves as a holistic metric of potential toxicity and disease outcome. CPTM quantitative toxicity is the measure of chemical features, biological phenotypic effects, and toxicokinetic properties of the ECs. For proof-of-concept, we subject ECs obtained from the Environmental Protection Agency's (EPA) database to the CPTM. We validated the CPTM toxicity predictions by correlating 'Zts' scores with known toxicity effects. We also confirmed the CPTM predictions with in-vitro, and in-vivo experiments. In in-vitro and zebrafish models, we showed that, mixtures of the motor oil and food additive 'Salpn' with endogenous nuclear receptor ligands such as Vitamin D3, dysregulated the nuclear receptors and key transcription pathways involved in Colorectal Cancer. Further, in a human patient derived cell organoid model, we found that a mixture of the widely used pesticides 'Tetramethrin' and 'Fenpropathrin' significantly impacts the population of patient derived pancreatic cancer cells and 3D organoid models to support rapid PDAC disease progression. The CPTM method is, to our knowledge, the first comprehensive toxico-physicochemical, and phenotypic bionetwork-based platform for efficient high-throughput screening of environmental chemical toxicity, mechanisms of action, and connection to disease outcomes.

Regulation of Chemosensitivity in Human Medulloblastoma Cells by p53 and the PI3 Kinase Signaling Pathway.

In medulloblastoma, p53 expression has been associated with chemoresistance and radiation resistance and with poor long-term outcomes in the p53-mutated sonic hedgehog, MYC-p53, and p53-positive medulloblastoma subgroups. We previously established a direct role for p53 in supporting drug resistance in medulloblastoma cells with high basal protein expression levels (D556 and DAOY). We now show that p53 genetic suppression in medulloblastoma cells with low basal p53 protein expression levels (D283 and UW228) significantly reduced drug responsiveness, suggesting opposing roles for low p53 protein expression levels. Mechanistically, the enhanced cell death by p53 knockdown in high-p53 cells was associated with an induction of mTOR/PI3K signaling. Both mTOR inhibition and p110α/PIK3CA induction confirmed these findings, which abrogated or accentuated the enhanced chemosensitivity response in D556 cells respectively while converse was seen in D283 cells. Co-treatment with G-actin-sequestering peptide, thymosin ß4 (Tß4), induced p-AKTS473 in both p53-high and p53-low cells, enhancing chemosensitivity in D556 cells while enhancing chemoresistance in D283 and UW228 cells. IMPLICATIONS: Collectively, we identified an unexpected role for the PI3K signaling in enhancing cell death in medulloblastoma cells with high basal p53 expression. These studies indicate that levels of p53 immunopositivity may serve as a diagnostic marker of chemotherapy resistance and for defining therapeutic targeting.

Paramagnetic Mn8Fe4-co-Polystyrene Nanobeads as a Potential T1-T2 Multimodal Magnetic Resonance Imaging Contrast Agent with In Vivo Studies.

In developing a cluster-nanocarrier design, as a magnetic resonance imaging contrast agent, we have investigated the enhanced relaxivity of a manganese and iron-oxo cluster grafted within a porous polystyrene nanobead with increased relaxivity due to a higher surface area. The synthesis of the cluster-nanocarrier for the cluster Mn8Fe4O12(O2CC6H4CH═CH2)16(H2O)4, cross-linked with polystyrene (the nanocarrier), under miniemulsion conditions is described. By including a branched hydrophobe, iso-octane, the resulting nanobeads are porous and ∼70 nm in diameter. The increased surface area of the nanobeads compared to nonporous nanobeads leads to an enhancement in relaxivity; r1 increases from 3.8 to 5.2 ± 0.1 mM-1 s-1, and r2 increases from 11.9 to 50.1 ± 4.8 mM-1 s-1, at 9.4 teslas, strengthening the potential for T1 and T2 imaging. Several metrics were used to assess stability, and the porosity produced no reduction in metal stability. Synchrotron X-ray fluorescence microscopy was used to demonstrate that the nanobeads remain intact in vivo. In depth, physicochemical characteristics were determined, including extensive pharmacokinetics, in vivo imaging, and systemic biodistribution analysis.

Optical and magnetic resonance imaging approaches for investigating the tumour microenvironment: state-of-the-art review and future trends.

The tumour microenvironment (TME) strongly influences tumorigenesis and metastasis. Two of the most characterized properties of the TME are acidosis and hypoxia, both of which are considered hallmarks of tumours as well as critical factors in response to anticancer treatments. Currently, various imaging approaches exist to measure acidosis and hypoxia in the TME, including magnetic resonance imaging (MRI), positron emission tomography and optical imaging. In this review, we will focus on the latest fluorescent-based methods for optical sensing of cell metabolism and MRI as diagnostic imaging tools applied both in vitro and in vivo. The primary emphasis will be on describing the current and future uses of systems that can measure intra- and extra-cellular pH and oxygen changes at high spatial and temporal resolution. In addition, the suitability of these approaches for mapping tumour heterogeneity, and assessing response or failure to therapeutics will also be covered.

Predicting new drug indications for prostate cancer: The integration of an in silico proteochemometric network pharmacology platform with patient-derived primary prostate cells.

BACKGROUND: Drug repurposing enables the discovery of potential cancer treatments using publically available data from over 4000 published Food and Drug Administration approved and experimental drugs. However, the ability to effectively evaluate the drug's efficacy remains a challenge. Impediments to broad applicability include inaccuracies in many of the computational drug-target algorithms and a lack of clinically relevant biologic modeling systems to validate the computational data for subsequent translation. METHODS: We have integrated our computational proteochemometric systems network pharmacology platform, DrugGenEx-Net, with primary, continuous cultures of conditionally reprogrammed (CR) normal and prostate cancer (PCa) cells derived from treatment-naive patients with primary PCa. RESULTS: Using the transcriptomic data from two matched pairs of benign and tumor-derived CR cells, we constructed drug networks to describe the biological perturbation associated with each prostate cell subtype at multiple levels of biological action. We prioritized the drugs by analyzing these networks for statistical coincidence with the drug action networks originating from known and predicted drug-protein targets. Prioritized drugs shared between the two patients' PCa cells included carfilzomib (CFZ), bortezomib (BTZ), sulforaphane, and phenethyl isothiocyanate. The effects of these compounds were then tested in the CR cells, in vitro. We observed that the IC50 values of the normal PCa CR cells for CFZ and BTZ were higher than their matched tumor CR cells. Transcriptomic analysis of CFZ-treated CR cells revealed that genes involved in cell proliferation, proteases, and downstream targets of serine proteases were inhibited while KLK7 and KLK8 were induced in the tumor-derived CR cells. CONCLUSIONS: Given that the drugs in the database are extremely well-characterized and that the patient-derived cells are easily scalable for high throughput drug screening, this combined in vitro and in silico approach may significantly advance personalized PCa treatment and for other cancer applications.

Inhibition of the mitochondrial citrate carrier, Slc25a1, reverts steatosis, glucose intolerance, and inflammation in preclinical models of NAFLD/NASH.

Nonalcoholic fatty liver disease (NAFLD) and its evolution to inflammatory steatohepatitis (NASH) are the most common causes of chronic liver damage and transplantation that are reaching epidemic proportions due to the upraising incidence of metabolic syndrome, obesity, and diabetes. Currently, there is no approved treatment for NASH. The mitochondrial citrate carrier, Slc25a1, has been proposed to play an important role in lipid metabolism, suggesting a potential role for this protein in the pathogenesis of this disease. Here, we show that Slc25a1 inhibition with a specific inhibitor compound, CTPI-2, halts salient alterations of NASH reverting steatosis, preventing the evolution to steatohepatitis, reducing inflammatory macrophage infiltration in the liver and adipose tissue, while starkly mitigating obesity induced by a high-fat diet. These effects are differentially recapitulated by a global ablation of one copy of the Slc25a1 gene or by a liver-targeted Slc25a1 knockout, which unravel dose-dependent and tissue-specific functions of this protein. Mechanistically, through citrate-dependent activities, Slc25a1 inhibition rewires the lipogenic program, blunts signaling from peroxisome proliferator-activated receptor gamma, a key regulator of glucose and lipid metabolism, and inhibits the expression of gluconeogenic genes. The combination of these activities leads not only to inhibition of lipid anabolic processes, but also to a normalization of hyperglycemia and glucose intolerance as well. In summary, our data show for the first time that Slc25a1 serves as an important player in the pathogenesis of fatty liver disease and thus, provides a potentially exploitable and novel therapeutic target.

The Sustained Induction of c-MYC Drives Nab-Paclitaxel Resistance in Primary Pancreatic Ductal Carcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. IMPLICATIONS: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.

Paramagnetic Clusters of Mn3(O2CCH3)6(Bpy)2 in Polyacrylamide Nanobeads as a New Design Approach to a T1- T2 Multimodal Magnetic Resonance Imaging Contrast Agent.

There is an increasing need for gadolinium-free magnetic resonance imaging (MRI) contrast agents, particularly for patients suffering from chronic kidney disease. Using a cluster-nanocarrier combination, we have identified a novel approach to the design of biomedical nanomaterials and report here the criteria for the cluster and the nanocarrier and the advantages of this combination. We have investigated the relaxivity of the following manganese oxo clusters: the parent cluster Mn3(O2CCH3)6(Bpy)2 (1) where Bpy = 2,2'-bipyridine and three new analogs, Mn3(O2CC6H4CH═CH2)6(Bpy)2 (2), Mn3(O2CC(CH3)═CH2)6(Bpy)2 (3), and Mn3O(O2CCH3)6(Pyr)2 (4) where Pyr = pyridine. The parent cluster, Mn3(O2CCH3)6(Bpy)2 (1), had impressive relaxivity ( r1 = 6.9 mM-1 s-1, r2 = 125 mM-1 s-1) and was found to be the most amenable for the synthesis of cluster-nanocarrier nanobeads. Using the inverse miniemulsion polymerization technique (1) in combination with the hydrophilic monomer acrylamide, we synthesized nanobeads (∼125 nm diameter) with homogeneously dispersed clusters within the polyacrylamide matrix (termed Mn3Bpy-PAm). The nanobeads were surface-modified by co-polymerization with an amine-functionalized monomer. This enabled various postsynthetic modifications, for example, to attach a near-IR dye, Cyanine7, as well as a targeting agent. When evaluated as a potential multimodal MRI contrast agent, high relaxivity and contrast were observed with r1 = 54.4 mM-1 s-1 and r2 = 144 mM-1 s-1, surpassing T1 relaxivity of clinically used Gd-DTPA chelates as well as comparable T2 relaxivity to iron oxide microspheres. Physicochemical properties, cellular uptake, and impacts on cell viability were also investigated.

Clinicopathologic and prognostic significance of LGR5, a cancer stem cell marker in patients with colorectal cancer.

AIM: To analyze the clinicopathologic and prognostic significance of Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a cancer stem cell marker expression in a cohort of colorectal cancer patients (CRC). PATIENTS & METHODS: A total of 76 formalin-fixed paraffin-embedded tissue blocks of primary or metastatic tumors from 49 CRC patients were collected for duration 2009-2015. LGR5 expression was assessed through immunohistochemical staining of a tissue microarray. RESULTS: LGR5 was significantly over expressed in CRC tissue samples and found to be a statistically significant independent prognostic marker for an improved overall survival. CONCLUSION: LGR5 expression was higher in colorectal cancer than in normal tissue. LGR5 was an independent prognostic marker for better clinical outcomes and might be used as a potential therapeutic target in CRCs.

The mitochondrial citrate carrier, SLC25A1, drives stemness and therapy resistance in non-small cell lung cancer.

Therapy resistance represents a clinical challenge for advanced non-small cell lung cancer (NSCLC), which still remains an incurable disease. There is growing evidence that cancer-initiating or cancer stem cells (CSCs) provide a reservoir of slow-growing dormant populations of cells with tumor-initiating and unlimited self-renewal ability that are left behind by conventional therapies reigniting post-therapy relapse and metastatic dissemination. The metabolic pathways required for the expansion of CSCs are incompletely defined, but their understanding will likely open new therapeutic opportunities. We show here that lung CSCs rely upon oxidative phosphorylation for energy production and survival through the activity of the mitochondrial citrate transporter, SLC25A1. We demonstrate that SLC25A1 plays a key role in maintaining the mitochondrial pool of citrate and redox balance in CSCs, whereas its inhibition leads to reactive oxygen species build-up thereby inhibiting the self-renewal capability of CSCs. Moreover, in different patient-derived tumors, resistance to cisplatin or to epidermal growth factor receptor (EGFR) inhibitor treatment is acquired through SLC25A1-mediated implementation of mitochondrial activity and induction of a stemness phenotype. Hence, a newly identified specific SLC25A1 inhibitor is synthetic lethal with cisplatin or with EGFR inhibitor co-treatment and restores antitumor responses to these agents in vitro and in animal models. These data have potential clinical implications in that they unravel a metabolic vulnerability of drug-resistant lung CSCs, identify a novel SLC25A1 inhibitor and, lastly, provide the first line of evidence that drugs, which block SLC25A1 activity, when employed in combination with selected conventional antitumor agents, lead to a therapeutic benefit.

Characterization of the effects of defined, multidimensional culture conditions on conditionally reprogrammed primary human prostate cells.

The inability to propagate human prostate epithelial cells indefinitely has historically presented a serious impediment to prostate cancer research. The conditionally reprogrammed cell (CRC) approach uses the combination of irradiated J2 mouse fibroblasts and a Rho kinase inhibitor such as Y27632 to support the continuous culture of cells derived from most epithelial tissues, including the prostate. Due to their rapid establishment and overall ease of use, CRCs are now widely used in a variety of basic and preclinical settings. In addition, CRCs were successfully used to clinically treat respiratory papillomatosis. Although both normal and tumor-derived prostate CRCs have been used to study the basic biology of prostate cancer and to test new therapies, certain limitations exist. We have previously reported that prostate CRCs form functional prostate glands when implanted under the mouse renal capsule. However in conventional culture, the prostate CRCs exist in an adult stem-like, transient amplifying state and consequently do not adequately recapitulate several important features of a differentiated prostate epithelium. To address these limitations, we previously described a transwell dish-based model that supported the culturing of prostate CRCs and the collection of cells and cell extracts for molecular and genetic analyses. Using normal and tumor-derived prostate CRCs, we describe the combined effects of the multi-dimensional transwell platform and defined culture media on prostate cellular proliferation, differentiation and signaling.

Protein drug target activation homogeneity in the face of intra-tumor heterogeneity: implications for precision medicine.

INTRODUCTION: Recent studies indicated tumors may be comprised of heterogeneous molecular subtypes and incongruent molecular portraits may emerge if different areas of the tumor are sampled. This study explored the impact of intra-tumoral heterogeneity in terms of activation/phosphorylation of FDA approved drug targets and downstream kinase substrates. MATERIAL AND METHODS: Two independent sets of liver metastases from colorectal cancer were used to evaluate protein kinase-driven signaling networks within different areas using laser capture microdissection and reverse phase protein array. RESULTS: Unsupervised hierarchical clustering analysis indicated that the signaling architecture and activation of the MAPK and AKT-mTOR pathways were consistently maintained within different regions of the same biopsy. Intra-patient variability of the MAPK and AKT-mTOR pathway were <1.06 fold change, while inter-patients variability reached fold change values of 5.01. CONCLUSIONS: Protein pathway activation mapping of enriched tumor cells obtained from different regions of the same tumor indicated consistency and robustness independent of the region sampled. This suggests a dominant protein pathway network may be activated in a high percentage of the tumor cell population. Given the genomic intra-tumoral variability, our data suggest that protein/phosphoprotein signaling measurements should be integrated with genomic analysis for precision medicine based analysis.

The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells.

Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB.

Thermoresponsive release of viable microfiltrated Circulating Tumor Cells (CTCs) for precision medicine applications.

Stimulus responsive release of Circulating Tumor Cells (CTCs), with high recovery rates from their capture platform, is highly desirable for off-chip analyses. Here, we present a temperature responsive polymer coating method to achieve both release as well as culture of viable CTCs captured from patient blood samples.

The induction of the p53 tumor suppressor protein bridges the apoptotic and autophagic signaling pathways to regulate cell death in prostate cancer cells.

The p53 tumor suppressor protein plays a crucial role in influencing cell fate decisions in response to cellular stress. As p53 elicits cell cycle arrest, senescence or apoptosis, the integrity of the p53 pathway is considered a key determinant of anti-tumor responses. p53 can also promote autophagy, however the role of p53-dependent autophagy in chemosensitivity is poorly understood. VMY-1-103 (VMY), a dansylated analog of purvalanol B, displays rapid and potent anti-tumor activities, however the pathways by which VMY works are not fully defined. Using established prostate cancer cell lines and novel conditionally reprogrammed cells (CRCs) derived from prostate cancer patients; we have defined the mechanisms of VMY-induced prostate cancer cell death. Herein, we show that the cytotoxic effects of VMY required a p53-dependent induction of autophagy, and that inhibition of autophagy abrogated VMY-induced cell death. Cancer cell lines harboring p53 missense mutations evaded VMY toxicity and treatment with a small molecule compound that restores p53 activity re-established VMY-induced cell death. The elucidation of the molecular mechanisms governing VMY-dependent cell death in cell lines, and importantly in CRCs, provides the rationale for clinical studies of VMY, alone or in combination with p53 reactivating compounds, in human prostate cancer.

Pilot phase I/II personalized therapy trial for metastatic colorectal cancer: evaluating the feasibility of protein pathway activation mapping for stratifying patients to therapy with imatinib and panitumumab.

This nonrandomized phase I/II trial assessed the efficacy/tolerability of imatinib plus panitumumab in patients affected by metastatic colorectal cancer (mCRC) after stratification to treatment by selection of activated imatinib drug targets using reverse-phase protein array (RPPA). mCRC patients presenting with a biopsiable liver metastasis were enrolled. Allocation to the experimental and control arms was established using functional pathway activation mapping of c-Kit, PDGFR, and c-Abl phosphorylation by RPPA. The experimental arm received run-in escalation therapy with imatinib followed by panitumumab. The control arm received panitumumab alone. Seven patients were enrolled in the study. For three of the seven patients, sequential pre- and post-treatment biopsies were used to evaluate the effect of the therapeutic compounds on the drug targets and substrates. A decrease in the activation level of the drug targets and downstream substrates was observed in two of three patients. Combination therapy increased the activation of the AKT-mTOR pathway and several receptor tyrosine kinases. This study proposes a novel methodology for stratifying patients to personalized treatment based on the activation level of the drug targets. This workflow provides the ability to monitor changes in the signaling pathways after the administration of targeted therapies and to identify compensatory mechanisms.