Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Sunitinib promotes myogenic regeneration and mitigates disease progression in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, muscle degenerative disease causing premature death of affected children. DMD is characterized by mutations in the dystrophin gene that result in a loss of the dystrophin protein. Loss of dystrophin causes an associated reduction in proteins of the dystrophin glycoprotein complex, leading to contraction-induced sarcolemmal weakening, muscle tearing, fibrotic infiltration and rounds of degeneration and failed regeneration affecting satellite cell populations. The α7ß1 integrin has been implicated in increasing myogenic capacity of satellite cells, therefore restoring muscle viability, increasing muscle force and preserving muscle function in dystrophic mouse models. In this study, we show that a Food and Drug Administration (FDA)-approved small molecule, Sunitinib, is a potent α7 integrin enhancer capable of promoting myogenic regeneration by stimulating satellite cell activation and increasing myofiber fusion. Sunitinib exerts its regenerative effects via transient inhibition of SHP-2 and subsequent activation of the STAT3 pathway. Treatment of mdx mice with Sunitinib demonstrated decreased membrane leakiness and damage owing to myofiber regeneration and enhanced support at the extracellular matrix. The decreased myofiber damage translated into a significant increase in muscle force production. This study identifies an already FDA-approved compound, Sunitinib, as a possible DMD therapeutic with the potential to treat other muscular dystrophies in which there is defective muscle repair.

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin. In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.

Targeting Estrogen Receptor Signaling with Fulvestrant Enhances Immune and Chemotherapy-Mediated Cytotoxicity of Human Lung Cancer.

PURPOSE: The conversion of tumor cells from an epithelial to a mesenchymal-like phenotype, via a process designated as the epithelial-mesenchymal transition (EMT), is known to mediate tumor resistance to a variety of cell death inducers, including cytotoxic effector immune cells. The goal of this study was to identify and potentially repurpose FDA-approved compounds capable of reducing mesenchymal features of human lung carcinoma cells, which could be used in combination with immunotherapies or chemotherapeutic strategies to improve clinical responses. EXPERIMENTAL DESIGN: In the current report, we have utilized a quantitative high-throughput screening (qHTS) of a pharmaceutical collection of more than 2,000 compounds to identify clinically approved drugs capable of augmenting the sensitivity of mesenchymal-like, lung cancer cells to immune- and chemotherapy-mediated lysis, both in vitro and in vivo RESULTS: The estrogen receptor antagonist fulvestrant was shown to reduce mesenchymal features of lung carcinoma cells, resulting in tumor sensitization to the cytotoxic effect of antigen-specific T cells, natural killer (NK) effector cells, and chemotherapy both in vivo and in vitro CONCLUSIONS: To our knowledge, this is the first report defining a potential role for estrogenic signaling in promoting tumor resistance to immune-mediated cytotoxicity and chemotherapy in lung cancer. Our data demonstrate a robust association between the acquisition of mesenchymal attributes, therapeutic resistance of lung carcinoma cells, and the expression of estrogen receptor 1 (ESR1), supporting further investigations on the role of estrogen signaling in lung cancer progression via the induction of EMT. Clin Cancer Res; 22(24); 6204-16. ©2016 AACR.

Small Molecule, NSC95397, Inhibits the CtBP1-Protein Partner Interaction and CtBP1-Mediated Transcriptional Repression.

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.