Loss of stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML.

The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely due to the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor GADD45A is implicated in poor clinical outcomes but its role in LSCs and AML pathogenesis is unknown. Here we define GADD45A as a key downstream target of LGR4 oncogenic signaling and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo and reduces levels of reactive oxygen species (ROS), accompanied by decreased response to ROS-associated genotoxic agents (e.g., ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype upon serial transplantation in mice. Our single-cell CITE-seq analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in AML patients. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Multifunctional Fluoropolymer-Engineered Magnetic Nanoparticles to Facilitate Blood-Brain Barrier Penetration and Effective Gene Silencing in Medulloblastoma.

Patients with brain cancers including medulloblastoma lack treatments that are effective long-term and without side effects. In this study, a multifunctional fluoropolymer-engineered iron oxide nanoparticle gene-therapeutic platform is presented to overcome these challenges. The fluoropolymers are designed and synthesized to incorporate various properties including robust anchoring moieties for efficient surface coating, cationic components to facilitate short interference RNA (siRNA) binding, and a fluorinated tail to ensure stability in serum. The blood-brain barrier (BBB) tailored system demonstrates enhanced BBB penetration, facilitates delivery of functionally active siRNA to medulloblastoma cells, and delivers a significant, almost complete block in protein expression within an in vitro extracellular acidic environment (pH 6.7) - as favored by most cancer cells. In vivo, it effectively crosses an intact BBB, provides contrast for magnetic resonance imaging (MRI), and delivers siRNA capable of slowing tumor growth without causing signs of toxicity - meaning it possesses a safe theranostic function. The pioneering methodology applied shows significant promise in the advancement of brain and tumor microenvironment-focused MRI-siRNA theranostics for the better treatment and diagnosis of medulloblastoma.

EGFR Targeting of Liposomal Doxorubicin Improves Recognition and Suppression of Non-Small Cell Lung Cancer.

Introduction: Despite improvements in chemotherapy and molecularly targeted therapies, the life expectancy of patients with advanced non-small cell lung cancer (NSCLC) remains less than 1 year. There is thus a major global need to advance new treatment strategies that are more effective for NSCLC. Drug delivery using liposomal particles has shown success at improving the biodistribution and bioavailability of chemotherapy. Nevertheless, liposomal drugs lack selectivity for the cancer cells and have a limited ability to penetrate the tumor site, which severely limits their therapeutic potential. Epidermal growth factor receptor (EGFR) is overexpressed in NSCLC tumors in about 80% of patients, thus representing a promising NSCLC-specific target for redirecting liposome-embedded chemotherapy to the tumor site. Methods: Herein, we investigated the targeting of PEGylated liposomal doxorubicin (Caelyx), a powerful off-the-shelf antitumoral liposomal drug, to EGFR as a therapeutic strategy to improve the specific delivery and intratumoral accumulation of chemotherapy in NSCLC. EGFR-targeting of Caelyx was enabled through its complexing with a polyethylene glycol (PEG)/EGFR bispecific antibody fragment. Tumor targeting and therapeutic potency of our treatment approach were investigated in vitro using a panel of NSCLC cell lines and 3D tumoroid models, and in vivo in a cell line-derived tumor xenograft model. Results: Combining Caelyx with our bispecific antibody generated uniform EGFR-targeted particles with improved binding and cytotoxic efficacy toward NSCLC cells. Effects were exclusive to cancer cells expressing EGFR, and increments in efficacy positively correlated with EGFR density on the cancer cell surface. The approach demonstrated increased penetration within 3D spheroids and was effective at targeting and suppressing the growth of NSCLC tumors in vivo while reducing drug delivery to the heart. Conclusion: EGFR targeting represents a successful approach to enhance the selectivity and therapeutic potency of liposomal chemotherapy toward NSCLC.

A Phase 1 Study of Intravenous EGFR-ErbituxEDVsMIT in Children with Solid or CNS Tumours Expressing Epidermal Growth Factor Receptor.

BACKGROUND: Recurrent or refractory solid and central nervous system (CNS) tumours in paediatric patients have limited treatment options and carry a poor prognosis. The EnGeneIC Dream Vector (EDV) is a novel nanocell designed to deliver cytotoxic medication directly to the tumour. The epidermal growth factor receptor is expressed in several CNS and solid tumours and is the target for bispecific antibodies attached to the EDV. OBJECTIVE: To assess the safety and tolerability of EGFR-Erbitux receptor EnGeneIC Dream Vector with mitoxantrone (EEDVsMit) in children with recurrent / refractory solid or CNS tumours expressing EGFR. PATIENTS AND METHODS: Patients aged 2-21 years with relapsed or refractory CNS and solid tumours, or radiologically diagnosed diffuse intrinsic pontine glioma (DIPG), were treated in this phase I open-label study of single agent EEDVsMit. Thirty-seven patients' tumours were screened for EGFR expression. EEDVsMit was administered twice weekly in the first cycle and weekly thereafter. Standard dose escalation with a rolling 6 design was employed. Dosing commenced at 5 × 108 EEDVsMit per dose and escalated to 5 × 109 EEDVsMit per dose. RESULTS: EGFR expression was detected in 12 (32%) of the paediatric tumours tested. Nine patients were enrolled and treated on the trial, including three patients with diffuse midline glioma. Overall, EEDVsMit was well tolerated, with no dose-limiting toxicities observed. The most common drug-related adverse events were grade 1-2 fever, nausea and vomiting, rash, lymphopaenia, and mildly deranged liver function tests. All patients had disease progression, including one patient who achieved a mixed response as the best response. CONCLUSIONS: EGFR-Erbitux receptor targeted EnGeneIC Dream Vector with mitoxantrone can be safely delivered in paediatric patients aged 2-21 years with solid or CNS tumours harbouring EGFR expression. The discovery of EGFR expression in a high proportion of paediatric gliomas means that EGFR may be useful as a target for other treatment strategies. Targeted therapeutic-loaded EDVs may be worth exploring further for their role in stimulating an anti-tumour immune response. GOV IDENTIFIER: NCT02687386.

Targeted delivery of polo-like kinase 1 siRNA nanoparticles using an EGFR-PEG bispecific antibody inhibits proliferation of high-risk neuroblastoma.

High-risk neuroblastoma has poor survival due to treatment failure and off-target side effects of therapy. Small molecule inhibitors have shown therapeutic efficacy at targeting oncogenic cell cycle dysregulators, such as polo-like kinase 1 (PLK1). However, their clinical success is limited by a lack of efficacy and specificity, causing off-target toxicity. Herein, we investigate a new treatment strategy whereby a bispecific antibody (BsAb) with dual recognition of methoxy polyethylene glycol (PEG) and a neuroblastoma cell-surface receptor, epidermal growth factor receptor (EGFR), is combined with a PEGylated small interfering RNA (siRNA) lipid nanoparticle, forming BsAb-nanoparticle RNA-interference complexes for targeted PLK1 inhibition against high-risk neuroblastoma. Therapeutic efficacy of this strategy was explored in neuroblastoma cell lines and a tumor xenograft model. Using ionizable lipid-based nanoparticles as a low-toxicity and clinically safe approach for siRNA delivery, we identified that their complexing with EGFR-PEG BsAb resulted in increases in cell targeting (1.2 to >4.5-fold) and PLK1 gene silencing (>2-fold) against EGFR+ high-risk neuroblastoma cells, and enhancements correlated with EGFR expression on the cells (r > 0.94). Through formulating nanoparticles with PEG-lipids ranging in diffusivity, we further identified a highly diffusible PEG-lipid which provided the most pronounced neuroblastoma cell binding, PLK1 silencing, and significantly reduced cancer growth in vitro in high-risk neuroblastoma cell cultures and in vivo in a tumor-xenograft mouse model of the disease. Together, this work provides an insight on the role of PEG-lipid diffusivity and EGFR targeting as potentially relevant variables influencing the therapeutic efficacy of siRNA nanoparticles in high-risk neuroblastoma.

An antibody fragment-decorated liposomal conjugate targets Philadelphia-like acute lymphoblastic leukemia.

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is an aggressive B-ALL malignancy associated with high rates of relapse and inferior survival rate. While targeted treatments against the cell surface proteins CD22 or CD19 have been transformative in the treatment of refractory B-ALL, patients may relapse due to antigen loss, necessitating targeting alternative antigens. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in half of Ph-like ALL cases conferring chemoresistance and enhancement of leukemia cell survival. Therefore, targeting CRLF2 may reduce the likelihood of relapse associated with antigen loss. We developed a CRLF2-targeting single-chain variable fragment modified by the fragment crystallizable region (CRLF2 scFv-Fc) conjugated to a drug maytansinoid 1 (DM1)-DOPC liposomal conjugate, creating homogeneous CRLF2-targeted liposomes (CRLF2-DM1 LIP). Cellular association and internalization studies in a Ph-like ALL cell line, MHH-CALL-4, compared to its lentivirally transduced CRLF2-knockdown counterpart (KD-CALL-4) revealed excellent CRLF2-targeting efficiency of CRLF2-DM1 LIP. Moreover, CRLF2-DM1 LIP showed selective association and internalization ex vivo using Ph-like ALL patient-derived xenograft (PDX) cells with minimal reactivity with non-target cells. Cell apoptosis assays demonstrated the CRLF2-dependent potency of CRLF2-DM1 LIP in Ph-like ALL cell lines. This study is the first to highlight the therapeutic potential of a CRLF2-directed scFv-Fc-liposomal conjugate for targeting Ph-like ALL.

3D Bioprintable Hydrogel with Tunable Stiffness for Exploring Cells Encapsulated in Matrices of Differing Stiffnesses.

In vitro cell models have undergone a shift from 2D models on glass slides to 3D models that better reflect the native 3D microenvironment. 3D bioprinting promises to progress the field by allowing the high-throughput production of reproducible cell-laden structures with high fidelity. The current stiffness range of printable matrices surrounding the cells that mimic the extracellular matrix environment remains limited. The work presented herein aims to expand the range of stiffnesses by utilizing a four-armed polyethylene glycol with maleimide-functionalized arms. The complementary cross-linkers comprised a matrix metalloprotease-degradable peptide and a four-armed thiolated polymer which were adjusted in ratio to tune the stiffness. The modularity of this system allows for a simple method of controlling stiffness and the addition of biological motifs. The application of this system in drop-on-demand printing is validated using MCF-7 cells, which were monitored for viability and proliferation. This study shows the potential of this system for the high-throughput investigation of the effects of stiffness and biological motif compositions in relation to cell behaviors.

Delivery of PEGylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia.

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.

Efficient delivery of Temozolomide using ultrasmall large-pore silica nanoparticles for glioblastoma.

The prognosis of brain cancers such as glioblastoma remains poor despite numerous advancements in the field of neuro-oncology. The presence of the blood brain barrier (BBB) along with the highly invasive and aggressive nature of glioblastoma presents a difficult challenge for developing effective therapies. Temozolomide (TMZ) is a first line agent used in the clinic for glioblastoma and it has been useful in increasing patient survival rates. However, TMZ suffers from issues related to its pharmacokinetics, such as a short plasma half-life (2 h), is subjected to P-gp efflux, and has limited extravasation from blood to brain (∼20%). It has been postulated that reducing its efflux and increasing glioblastoma tissue exposure to TMZ could prove useful in treating glioblastoma and preventing tumour recurrence. Herein, ultra-small, large pore silica nanoparticles (USLP) have been loaded with TMZ, surface PEGlyated to reduce efflux and decorated with the cascade targeting protein lactoferrin for efficient uptake across the BBB and into glioblastoma. Our results demonstrate that USLP improves permeability of BBB in vitro as evidenced using a transwell model which mimics endothelial tight junctions with permeation being enhanced using PEGylated particles. Data from TMZ loaded USLP in vitro transwell BBB model also suggests that the USLP formulations can significantly reduce the efflux ratio of TMZ. In vitro apoptosis studies on glioblastoma cell lines U87 and GL261 were conducted which showed an improvement in TMZ induced glioblastoma apoptosis with USLP formulations compared to pure TMZ. Finally, a proof-of-concept preclinical mouse study demonstrated that when given intravenously at 50 mg/kg, USLP particles showed accumulation in the brain within a few hours without any obvious pathophysiological changes in vital organs as assessed via histology. Overall, the data suggests our innovative delivery system is efficient in extravasation from blood and permeating the BBB and has potential to improve efficacy of TMZ in glioblastoma therapy.

From cells to organoids: The evolution of blood-brain barrier technology for modelling drug delivery in brain cancer.

Brain cancer remains the deadliest cancer. The blood-brain barrier (BBB) is impenetrable to most drugs and is a complex 3D network of multiple cell types including endothelial cells, astrocytes, and pericytes. In brain cancers, the BBB becomes disrupted during tumor progression and forms the blood-brain tumor barrier (BBTB). To advance therapeutic development, there is a critical need for physiologically relevant BBB in vitro models. 3D cell systems are emerging as valuable preclinical models to accelerate discoveries for diseases. Given the versatility and capability of 3D cell models, their potential for modelling the BBB and BBTB is reviewed. Technological advances of BBB models and challenges of in vitro modelling the BBTB, and application of these models as tools for assessing therapeutics and nano drug delivery, are discussed. Quantitative, in vitro BBB models that are predictive of effective brain cancer therapies will be invaluable for accelerating advancing new treatments to the clinic.

Electrostatic Assembly of Multiarm PEG-Based Hydrogels as Extracellular Matrix Mimics: Cell Response in the Presence and Absence of RGD Cell Adhesive Ligands.

Synthetic hydrogels have been used widely as extracellular matrix (ECM) mimics due to the ability to control and mimic physical and biochemical cues observed in natural ECM proteins such as collagen, laminin, and fibronectin. Most synthetic hydrogels are formed via covalent bonding resulting in slow gelation which is incompatible with drop-on-demand 3D bioprinting of cells and injectable hydrogels for therapeutic delivery. Herein, we developed an electrostatically crosslinked PEG-based hydrogel system for creating high-throughput 3D in vitro models using synthetic hydrogels to mimic the ECM cancer environment. A 3-arm PEG-based polymer backbone was first modified with either permanent cationic charged moieties (2-(methacryloyloxy)ethyl trimethylammonium) or permanent anionic charged moieties (3-sulfopropyl methacrylate potassium salt). The resulting charged polymers can be conjugated further with various amounts of cell adhesive RGD motifs (0, 25, 75, and 98%) to study the influences of RGD motifs on breast cancer (MCF-7) spheroid formation. Formation, stability, and mechanical properties of hydrogels were tested with, and without, RGD to evaluate the cellular response to material parameters in a 3D environment. The hydrogels can be degraded in the presence of salts at room temperature by breaking the interaction of oppositely charged polymer chains. MCF-7 cells could be released with high viability through brief exposure to NaCl solution. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promoting cell proliferation in the stiffer hydrogel. Flow cytometry characterization demonstrated that embedded MCF-7 cells proliferate better in a softer (60 Pa) 3D hydrogel environment compared to those that are stiffer (1160 Pa). As the stiffness increases, the RGD motif plays a role in promoting cell proliferation in the stiffer hydrogel. Additionally, cell viability was not impacted by the tested hydrogel stiffness range between 60 to 1160 Pa. Taken together, this PEG-based tuneable hydrogel system shows great promise as a 3D ECM mimic of cancer extracellular environments with controllable biophysical and biochemical properties. The ease of gelation and dissolution through salt concentration provides a way to quickly harvest cells for further analysis at any given time of interest without compromising cell viability.

Tuning the Mechanical Properties of Multiarm RAFT-Based Block Copolyelectrolyte Hydrogels via Ionic Cross-Linking for 3D Cell Cultures.

Hydrogels that serve as native extracellular matrix (ECM) mimics are typically naturally derived hydrogels that are physically cross-linked via ionic interactions. This means rapid gelation of synthetic polymers, which give control over the chemical and physical cues in hydrogel formation. Herein, we combine the best of both systems by developing a synthetic hydrogel with ionic cross-linking of block copolyelectrolytes to rapidly create hydrogels. Reversible addition-fragmentation chain-transfer (RAFT) polymerization was used to synthesize oppositely charged polyelectrolyte molecules and, in turn, modulate the mechanical property of stiffness. The mechanical stiffness of a range of 900-3500 Pa was tuned by varying the number of charged ionic groups, the length of the polymer arms, and the polymer concentration. We demonstrate the synthetic polyelectrolyte hydrogel as an ECM mimic for three-dimensional (3D) in vitro cell models using MCF-7 breast cancer cells.

ßIII-tubulin suppression enhances the activity of Amuvatinib to inhibit cell proliferation in c-Met positive non-small cell lung cancer cells.

Non-Small Cell Lung Carcinoma (NSCLC) remains a leading cause of cancer death. Resistance to therapy is a significant problem, highlighting the need to find new ways of sensitising tumour cells to therapeutic agents. ßIII-tubulin is associated with aggressive tumours and chemotherapy resistance in a range of cancers including NSCLC. ßIII-tubulin expression has been shown to impact kinase signalling in NSCLC cells. Here, we sought to exploit this interaction by identifying co-activity between ßIII-tubulin suppression and small-molecule kinase inhibitors. To achieve this, a forced-genetics approach combined with a high-throughput drug screen was used. We show that activity of the multi-kinase inhibitor Amuvatinib (MP-470) is enhanced by ßIII-tubulin suppression in independent NSCLC cell lines. We also show that this compound significantly inhibits cell proliferation among ßIII-tubulin knockdown cells expressing the receptor tyrosine kinase c-Met. Together, our results highlight that ßIII-tubulin suppression combined with targeting specific receptor tyrosine kinases may represent a novel therapeutic approach for otherwise difficult-to-treat lung carcinomas.

A high-throughput 3D bioprinted cancer cell migration and invasion model with versatile and broad biological applicability.

Understanding the underlying mechanisms of migration and metastasis is a key focus of cancer research. There is an urgent need to develop in vitro 3D tumor models that can mimic physiological cell-cell and cell-extracellular matrix interactions, with high reproducibility and that are suitable for high throughput (HTP) drug screening. Here, we developed a HTP 3D bioprinted migration model using a bespoke drop-on-demand bioprinting platform. This HTP platform coupled with tunable hydrogel systems enables (i) the rapid encapsulation of cancer cells within in vivo tumor mimicking matrices, (ii) in situ and real-time measurement of cell movement, (iii) detailed molecular analysis for the study of mechanisms underlying cell migration and invasion, and (iv) the identification of novel therapeutic options. This work demonstrates that this HTP 3D bioprinted cell migration platform has broad applications across quantitative cell and cancer biology as well as drug screening.

How can we use the endocytosis pathways to design nanoparticle drug-delivery vehicles to target cancer cells over healthy cells?

Targeted drug delivery in cancer typically focuses on maximising the endocytosis of drugs into the diseased cells. However, there has been less focus on exploiting the differences in the endocytosis pathways of cancer cells versus non-cancer cells. An understanding of the endocytosis pathways in both cancer and non-cancer cells allows for the design of nanoparticles to deliver drugs to cancer cells whilst restricting healthy cells from taking up anticancer drugs, thus efficiently killing the cancer cells. Herein we compare the differences in the endocytosis pathways of cancer and healthy cells. Second, we highlight the importance of the physicochemical properties of nanoparticles (size, shape, stiffness, and surface chemistry) on cellular uptake and how they can be adjusted to selectively target the dominated endocytosis pathway of cancer cells over healthy cells and to deliver anticancer drug to the target cells. The review generates new thought in the design of cancer-selective nanoparticles based on the endocytosis pathways.

Advances in 3D Bioprinting for Cancer Biology and Precision Medicine: From Matrix Design to Application.

The tumor microenvironment is highly complex owing to its heterogeneous composition and dynamic nature. This makes tumors difficult to replicate using traditional 2D cell culture models that are frequently used for studying tumor biology and drug screening. This often leads to poor translation of results between in vitro and in vivo and is reflected in the extremely low success rates of new candidate drugs delivered to the clinic. Therefore, there has been intense interest in developing 3D tumor models in the laboratory that are representative of the in vivo tumor microenvironment and patient samples. 3D bioprinting is an emerging technology that enables the biofabrication of structures with the virtue of providing accurate control over distribution of cells, biological molecules, and matrix scaffolding. This technology has the potential to bridge the gap between in vitro and in vivo by closely recapitulating the tumor microenvironment. Here, a brief overview of the tumor microenvironment is provided and key considerations in biofabrication of tumor models are discussed. Bioprinting techniques and choice of bioinks for both natural and synthetic polymers are also outlined. Lastly, current bioprinted tumor models are reviewed and the perspectives of how clinical applications can greatly benefit from 3D bioprinting technologies are offered.

Challenges and opportunities to penetrate the blood-brain barrier for brain cancer therapy.

Despite significant advances in research, the prognosis for both primary and secondary brain cancers remains poor. The blood-brain barrier (BBB) is a complex and unique semi-permeable membrane that serves as a protective structure to maintain homeostasis within the brain. However, it presents a significant challenge for the delivery of therapeutics into the brain and tumor. Some brain tumors are known to compromise BBB integrity, producing a highly heterogeneous vasculature known as the blood-tumor-barrier (BTB). Identifying strategies to bypass these obstacles to improve the penetrability of anticancer therapeutics has been the focus of research in this area. In this review, we discuss the strategies that have been investigated to evade or alter the cellular and molecular barriers of both the BBB and the BTB and detail the methods currently under preclinical or clinical investigation, including molecular, biological, and physical processes to overcome the BBB or BTB. Increased understanding of the BBB and BTB and the current methods of overcoming these barriers will enable the development of new and more effective treatment strategies for brain tumors.

High temporal resolution RNA-seq time course data reveals widespread synchronous activation between mammalian lncRNAs and neighboring protein-coding genes.

The advent of massively parallel sequencing revealed extensive transcription beyond protein-coding genes, identifying tens of thousands of long noncoding RNAs (lncRNAs). Selected functional examples raised the possibility that lncRNAs, as a class, may maintain broad regulatory roles. Expression of lncRNAs is strongly linked with adjacent protein-coding gene expression, suggesting potential cis-regulatory functions. A more detailed understanding of these regulatory roles may be obtained through careful examination of the precise timing of lncRNA expression relative to adjacent protein-coding genes. Despite the diversity of reported lncRNA regulatory mechanisms, where causal cis-regulatory relationships exist, lncRNA transcription is expected to precede changes in target gene expression. Using a high temporal resolution RNA-seq time course, we profiled the expression dynamics of several thousand lncRNAs and protein-coding genes in synchronized, transitioning human cells. Our findings reveal that lncRNAs are expressed synchronously with adjacent protein-coding genes. Analysis of lipopolysaccharide-activated mouse dendritic cells revealed the same temporal relationship observed in transitioning human cells. Our findings suggest broad-scale cis-regulatory roles for lncRNAs are not common. The strong association between lncRNAs and adjacent genes may instead indicate an origin as transcriptional by-products from active protein-coding gene promoters and enhancers.

ßIII-Tubulin Gene Regulation in Health and Disease.

Microtubule proteins form a dynamic component of the cytoskeleton, and play key roles in cellular processes, such as vesicular transport, cell motility and mitosis. Expression of microtubule proteins are often dysregulated in cancer. In particular, the microtubule protein ßIII-tubulin, encoded by the TUBB3 gene, is aberrantly expressed in a range of epithelial tumours and is associated with drug resistance and aggressive disease. In normal cells, TUBB3 expression is tightly restricted, and is found almost exclusively in neuronal and testicular tissues. Understanding the mechanisms that control TUBB3 expression, both in cancer, mature and developing tissues will help to unravel the basic biology of the protein, its role in cancer, and may ultimately lead to the development of new therapeutic approaches to target this protein. This review is devoted to the transcriptional and posttranscriptional regulation of TUBB3 in normal and cancerous tissue.