Cancer Biology

Genome-wide gene expression analysis is a commonly used method to quantitatively examine the transcriptional signature of any tissue or cell state. Standard bulk cell RNA sequencing (RNA-seq) quantifies RNAs in the cells of the tissue type of interest through massive parallel sequencing of cDNA synthesized from the cellular RNA. The subsequent analysis of global RNA expression and normalization of RNA expression levels between two or more samples generally assumes that cells from all samples produce equivalent amounts of RNA per cell. This assumption may be invalid in cells where MYC or MYCN expression levels are markedly different and thus, overall mRNA expression per cell is altered. Here, we describe an approach for RNA-seq analysis of MYCN-amplified neuroblastoma cells during treatment with retinoic acid, which causes dramatic downregulation of MYCN expression and induces growth arrest and differentiation of the cells. Our procedure employs spiked-in RNA standards added in ratio to the number of cells in each sample prior to RNA extraction. In the analysis of differential gene expression, the expression level of each gene is standardized to the spiked-in RNA standard to accurately assess gene expression levels per cell in conditions of high and low MYCN expression. Our protocol thus provides a step-by-step experimental approach for normalizing RNA-seq expression data on a per-cell-number basis, allowing accurate assessment of differential gene expression in cells expressing markedly different levels of MYC or MYCN.

The cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint assay (ITDRF _CETSA) have been introduced as powerful tools for investigating target engagement by measuring ligand-triggered thermodynamic stabilization of cellular target proteins. Yet, these techniques have rarely been used to evaluate the thermal stability of RNA-binding proteins (RBPs) when exposed to ligands. Here, we present an adjusted approach using CETSA and ITDRF_CETSA to determine the interaction between enasidenib and RBM45. Our assay is sensitive and time-efficient and can potentially be adapted for studying the interactions of RBM45 protein with other potential candidates.

Several assays have been developed to monitor the in vitro catalytic activity of Hedgehog acyltransferase (Hhat), an enzyme critical to the Hedgehog signaling pathway in cells. However, the majority of these previously reported assays involve radioactive fatty acyl donor substrates, multiple steps to achieve product readout, or specialized equipment. To increase safety, efficiency, and convenience, we developed a direct, fluorescent in vitro assay to monitor Hhat activity. Our assay utilizes purified Hhat, a fluorescently labeled fatty acyl-CoA donor substrate, and a Sonic hedgehog (Shh) peptide recipient substrate sufficient for fatty acylation. The protocol is a straightforward process that yields direct readout of fatty acylated Shh peptide via fluorescence detection of the transferred fatty acyl group.

Graphical abstract

Graphical abstract adapted from Schonbrun and Resh (2022)

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein and transcriptional regulator implicated in chromatin dynamics and cancer development. Several BRD4 isoforms have been detected in humans with the long isoform (BRD4-L, aa 1-1,362) playing a tumor-suppressive role and a major short isoform (BRD4-S, aa 1-722) having oncogenic activity in breast cancer development. In vivo demonstration of the opposing functions of BRD4 protein isoforms requires development of mouse models, particularly transgenic mice conditionally expressing human BRD4-L or BRD4-S, which can be selectively induced in different mouse tissues in a spatiotemporal-specific manner. Here, we detail the procedures used to genotype transgenic mouse strains developed to define the effects of conditional human BRD4 isoform expression on polyomavirus middle T antigen (PyMT)-induced mouse mammary tumor growth, and the key steps for Western blot detection of BRD4 protein isoforms in those tumors and in cultured cells. With this protocol as a guide, interpretation of BRD4 isoform functions becomes more feasible and expandable to various biological settings. Adequate tracking of BRD4 isoform distributions in vivo and in vitro is key to understanding their biological roles, as well as avoiding misinterpretation of their functions due to improper use of experimental procedures that fail to detect their spatial and temporal distributions.

Graphic abstract:

Targeting hard-to-drug proteins, such as proteins functioning by protein-protein interactions (PPIs) with small molecules, is difficult because of the lack of well-defined pockets. Fragment or computational-based methods are usually employed for the discovery of such compounds, but no generic method is available to quickly identify small molecules interfering with PPIs. Here, we provide a protocol describing a generic method to discover small molecules inhibiting the interaction between an intracellular antibody and its target, in particular for proteins that are hard to make in recombinant form. This protocol reports a versatile and generic method that can be applied to any target/intracellular antibody. Because it is a cell-based assay, it identifies chemical matters that are already displaying advantageous cell permeability properties.

Graphic abstract:

Cell-based intracellular antibody-guided small molecule screening.

Chemoresistance, the ability of cancer cells to overcome therapeutic interventions, is an area of active research. Studies on intrinsic and acquired chemoresistance have partly succeeded in elucidating some of the molecular mechanisms in this elusive phenomenon. Hence, drug-resistant cellular models are routinely developed and used to mimic the clinical scenario in-vitro. In an attempt to identify the underlying molecular mechanisms that allow ovarian cancer cells to gradually acquire chemoresistance, we have developed isogenic cellular models of cisplatin and paclitaxel resistance (singularly and in combination) over six months, using a clinically relevant modified pulse method. These models serve as important tools to investigate the underlying molecular players, modulation in genetics, epigenetics, and relevant signaling pathways, as well as to understand the role of drug detoxification and drug influx-efflux pathways in development of resistance. These models can also be used as screening tools for new therapeutic molecules. Additionally, repurposing therapeutic agents approved for diseases other than cancer have gained significant attention in improving cancer therapy. To investigate the effect of metformin on acquirement of chemoresistance, we have also developed a combinatorial model of metformin and platinum-taxol, using two different strategies. All these models were subsequently used to study modulation in receptor tyrosine kinase pathways, cancer stem cell functionalities, autophagy, metastasis, metabolic signatures, and various biological processes during development of chemoresistance. Herein, we outline the protocols used for developing these intricate resistant cellular models.

Graphic abstract:

Schematic of the step-wise development of cellular chemoresistant model. Schema illustrating the modified pulse method for the development of individual and combinatorial resistant models. Two different cell lines (A2780 and OAW42) were exposed to drug treatment (either alone or in various combinations) of one concentration for three consecutive cycles (3×). Furthermore, the surviving cells were sub-cultured subsequently with increasing drug concentrations. After every treatment cycle, 50% of the cells were cryo-preserved for further experiments. Additionally, to check for the development of chemoresistance and to assess the changes in cell cycle during resistance development, cell viability assays and flow cytometry were performed.

ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) is a powerful method to evaluate chromatin accessibility and nucleosome positioning at a genome-wide scale. This assay uses a hyperactive Tn5 transposase, to simultaneously cut open chromatin and insert adapter sequences. After sequencing, the reads generated through this technique are generally indicative of transcriptional regulatory elements that are located in accessible chromatin. This method was originally developed by Buenrostro et al. (2013), and since then it has been improved by the same authors several times, until their last update called OMNI ATAC-seq (Corces et al., 2017). Here, we describe an ATAC-seq protocol based on the OMNI-ATAC method, with a special focus on the initial steps of thawing cryopreserved cells, and the final steps of library purification using magnetic beads. This protocol can be of interest for laboratories working in a fast-paced environment.

Graphic abstract:

Flowchart of the protocol

In the cell, the thermodynamic stability of a protein – and hence its biological activity – can change dramatically as a result of perturbations in its amino acid sequence and the concentration of stabilizing ligands. This interplay is particularly evident in zinc-binding transcription factors such as the p53 tumor suppressor, whose DNA-binding activity can critically depend on levels of intracellular zinc as well as point mutations that alter either metal binding or folding stability. Separate protocols exist for determining a protein’s metal affinity and its folding free energy. These properties, however, are intimately connected, and a technique is needed to integrate these measurements. Our protocols employ common non-fluorescent and fluorescent zinc chelators to control and report on free Zn²⁺ concentration, respectively, combined with biophysical assays of full-length human p53 and its DNA-binding domain. Fitting the data to equations that contain stability and metal-binding terms results in a more complete picture of how metal-dependent proteins can lose and gain DNA-binding function in a range of physiological conditions.

Graphic abstract:

Figure 1. Raising intracellular zinc can restore tumor-suppressing function to p53 that has been unfolded by missense mutation or cellular conditions

Once thought to be a mere consequence of the state of a cell, intermediary metabolism is now recognized as a key regulator of mammalian cell fate and function. In addition, cell metabolism is often disturbed in malignancies such as cancer, and targeting metabolic pathways can provide new therapeutic options. Cell metabolism is mostly studied in cell cultures in vitro, using techniques such as metabolomics, stable isotope tracing, and biochemical assays. Increasing evidence however shows that the metabolic profile of cells is highly dependent on the microenvironment, and metabolic vulnerabilities identified in vitro do not always translate to in vivo settings. Here, we provide a detailed protocol on how to perform in vivo stable isotope tracing in leukemia cells in mice, focusing on glutamine metabolism in acute myeloid leukemia (AML) cells. This method allows studying the metabolic profile of leukemia cells in their native bone marrow niche.

This protocol illustrates a pipeline for modeling the nonlinear behavior of intracellular signaling pathways. At fixed spatial points, nonlinear signaling dynamics are described by ordinary differential equations (ODEs). At constant parameters, these ODEs may have multiple attractors, such as multiple steady states or limit cycles. Standard optimization procedures fine-tune the parameters for the system trajectories localized within the basin of attraction of only one attractor, usually a stable steady state. The suggested protocol samples the parameter space and captures the overall dynamic behavior by analyzing the number and stability of steady states and the shapes of the assembly of nullclines, which are determined as projections of quasi-steady-state trajectories into different 2D spaces of system variables. Our pipeline allows identifying main qualitative features of the model behavior, perform bifurcation analysis, and determine the borders separating the different dynamical regimes within the assembly of 2D parametric planes. Partial differential equation (PDE) systems describing the nonlinear spatiotemporal behavior are derived by coupling fixed point dynamics with species diffusion.