Alternative splicing in prostate cancer progression and therapeutic resistance.

Prostate cancer (CaP) remains the second leading cause of cancer deaths in western men. CaP mortality results from diverse molecular mechanisms that mediate resistance to the standard of care treatments for metastatic disease. Recently, alternative splicing has been recognized as a hallmark of CaP aggressiveness. Alternative splicing events cause treatment resistance and aggressive CaP behavior and are determinants of the emergence of the two major types of late-stage treatment-resistant CaP, namely castration-resistant CaP (CRPC) and neuroendocrine CaP (NEPC). Here, we review recent multi-omics data that are uncovering the complicated landscape of alternative splicing events during CaP progression and the impact that different gene transcript isoforms can have on CaP cell biology and behavior. We discuss renewed insights in the molecular machinery by which alternative splicing occurs and contributes to the failure of systemic CaP therapies. The potential for alternative splicing events to serve as diagnostic markers and/or therapeutic targets is explored. We conclude by considering current challenges and promises associated with splicing-modulating therapies, and their potential for clinical translation into CaP patient care.

Prostate Cancer Progression Relies on the Mitotic Kinase Citron Kinase.

Prostate cancer remains the second leading cause of cancer death in men in Western cultures. A deeper understanding of the mechanisms by which prostate cancer cells divide to support tumor growth could help devise strategies to overcome treatment resistance and improve survival. Here, we identified that the mitotic AGC family protein kinase citron kinase (CIT) is a pivotal regulator of prostate cancer growth that mediates prostate cancer cell interphase progression. Increased CIT expression correlated with prostate cancer growth induction and aggressive prostate cancer progression, and CIT was overexpressed in prostate cancer compared with benign prostate tissue. CIT overexpression was controlled by an E2F2-Skp2-p27 signaling axis and conferred resistance to androgen-targeted treatment strategies. The effects of CIT relied entirely on its kinase activity. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of prostate cancer progression and treatment resistance but did not affect benign epithelial prostate cells or nonprostatic normal cells, indicating a potential therapeutic window for CIT inhibition. CIT kinase activity was identified as druggable and was potently inhibited by the multikinase inhibitor OTS-167, which decreased the proliferation of treatment-resistant prostate cancer cells and patient-derived organoids. Isolation of the in vivo CIT substrates identified proteins involved in diverse cellular functions ranging from proliferation to alternative splicing events that are enriched in treatment-resistant prostate cancer. These findings provide insights into the regulation of aggressive prostate cancer cell behavior by CIT and identify CIT as a functionally diverse and druggable driver of prostate cancer progression. SIGNIFICANCE: The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008.

Valproic Acid-Induced Upregulation of Multidrug Efflux Transporter ABCG2/BCRP via PPARα-Dependent Mechanism in Human Brain Endothelial Cells.

Despite the progress made in the development of new antiepileptic drugs (AEDs), poor response to them is a rising concern in epilepsy treatment. Of several hypotheses explaining AED treatment failure, the most promising theory is the overexpression of multidrug transporters belonging to ATP-binding cassette (ABC) transporter family at blood-brain barrier. Previous data show that AEDs themselves can induce these transporters, in turn affecting their own brain bioavailability. Presently, this induction and the underlying regulatory mechanism involved at human blood-brain barrier is not well elucidated. Herein, we sought to explore the effect of most prescribed first- and second-line AEDs on multidrug transporters in human cerebral microvascular endothelial cells, hCMEC/D3. Our work demonstrated that exposure of these cells to valproic acid (VPA) induced mRNA, protein, and functional activity of breast cancer resistance protein (BCRP/ABCG2). On examining the substrate interaction status of AEDs with BCRP, VPA, phenytoin, and lamotrigine were found to be potential BCRP substrates. Furthermore, we observed that siRNA-mediated knockdown of peroxisome proliferator-activated receptor alpha (PPARα) or use of PPARα antagonist, resulted in attenuation of VPA-induced BCRP expression and transporter activity. VPA was found to increase PPARα expression and trigger its translocation from cytoplasm to nucleus. Findings from chromatin immunoprecipitation and luciferase assays showed that VPA enhances the binding of PPARα to its response element in the ABCG2 promoter, resulting in elevated ABCG2 transcriptional activity. Taken together, these in vitro findings highlight PPARα as the potential molecular target to prevent VPA-mediated BCRP induction, which may have important implications in VPA pharmacoresistance. SIGNIFICANCE STATEMENT: Induction of multidrug transporters at blood-brain barrier can largely affect the bioavailability of the substrate antiepileptic drugs in the brains of patients with epilepsy, thus affecting their therapeutic efficacy. The present study reports a mechanistic pathway of breast cancer resistance protein (BCRP/ABCG2) upregulation by valproic acid in human brain endothelial cells via peroxisome proliferator-activated receptor alpha involvement, thereby providing a potential strategy to prevent valproic acid pharmacoresistance in epilepsy.

Multidrug efflux transporter ABCG2: expression and regulation.

The adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) was originally discovered in a multidrug-resistant breast cancer cell line. Studies in the past have expanded the understanding of its role in physiology, disease pathology and drug resistance. With a widely distributed expression across different cell types, ABCG2 plays a central role in ATP-dependent efflux of a vast range of endogenous and exogenous molecules, thereby maintaining cellular homeostasis and providing tissue protection against xenobiotic insults. However, ABCG2 expression is subjected to alterations under various pathophysiological conditions such as inflammation, infection, tissue injury, disease pathology and in response to xenobiotics and endobiotics. These changes may interfere with the bioavailability of therapeutic substrate drugs conferring drug resistance and in certain cases worsen the pathophysiological state aggravating its severity. Considering the crucial role of ABCG2 in normal physiology, therapeutic interventions directly targeting the transporter function may produce serious side effects. Therefore, modulation of transporter regulation instead of inhibiting the transporter itself will allow subtle changes in ABCG2 activity. This requires a thorough comprehension of diverse factors and complex signaling pathways (Kinases, Wnt/ß-catenin, Sonic hedgehog) operating at multiple regulatory levels dictating ABCG2 expression and activity. This review features a background on the physiological role of transporter, factors that modulate ABCG2 levels and highlights various signaling pathways, molecular mechanisms and genetic polymorphisms in ABCG2 regulation. This understanding will aid in identifying potential molecular targets for therapeutic interventions to overcome ABCG2-mediated multidrug resistance (MDR) and to manage ABCG2-related pathophysiology.

Exploring the Carbamazepine Interaction with Human Pregnane X Receptor and Effect on ABCC2 Using in Vitro and in Silico Approach.

PURPOSE: Over expression of ATP-binding cassette transporters is considered one of the major reasons for non-responsiveness to antiepileptic drugs. Carbamazepine (CBZ), one of first line antiepileptic drug is known to influence ABCC2 expression but its exact molecular mechanism is unknown. METHODS: We investigated the effect of CBZ on expression of ABCC2 and pregnane X receptor (PXR) in HepG2 cell line and compared with hyperforin (agonist of PXR) and ketoconazole (antagonist of PXR) through realtime PCR and western blot assay. Involvement of PXR was demonstrated through nuclear translocation and RNA interference and related effect of CBZ on ABCC2 through functional activity assay. Molecular docking and dynamic simulation approach was used to understand the interaction of CBZ with PXR. RESULTS: CBZ and hyperforin increased the PXR and ABCC2 expression whereas reversed when present it in combination with ketoconazole. Experiments confirmed CBZ induced ABCC2 expression is PXR dependent. Molecular dynamic (MD) simulation and in vitro experiment indicated possibility of CBZ to be PXR agonist and PXR residue Gln285 to be important for CBZ-PXR interaction. CONCLUSIONS: CBZ alters the functional activity of ABCC2 through PXR, which in turn can interfere with therapy. Mutational analysis of residues revealed the importance of Gln285 in ligand interaction.