Importins and exportins as therapeutic targets in cancer

Abstract

The nuclear transport proteins, importins and exportins (karyopherin-β proteins), may play an important role in cancer by transporting key mediators of oncogenesis across the nuclear membrane in cancer cells. During nucleocytoplasmic transport of tumor suppressor proteins and cell cycle regulators during the processing of these proteins, aberrant cellular growth signaling and inactivation of apoptosis can occur, both critical to growth and development of tumors. Karyopherin-β proteins bind to these cargo proteins and RanGTP for active transport across the nuclear membrane through the nuclear pore complex. Importins and exportins are overexpressed in multiple tumors including melanoma, pancreatic, breast, colon, gastric, prostate, esophageal, lung cancer, and lymphomas. Furthermore, some of the karyopherin-β proteins such as exportin-1 have been implicated in drug resistance in cancer. Importin and exportin inhibitors are being considered as therapeutic targets against cancer and have shown preclinical anticancer activity. Moreover, synergistic activity has been observed with various chemotherapeutic and targeted agents. However, clinical development of the exportin-1 inhibitor leptomycin B was stopped due to adverse events, including vomiting, anorexia, and dehydration. Selinexor, a selective nuclear export inhibitor, is being tested in multiple clinical trials both as a single agent and in combination with chemotherapy. Selinexor has demonstrated clinical activity in multiple cancers, especially acute myelogenous leukemia and multiple myeloma. The roles of other importin and exportin inhibitors still need to be investigated clinically. Targeting the key mediators of nucleocytoplasmic transport in cancer cells represents a novel strategy in cancer intervention with the potential to significantly affect outcomes.

Introduction

Compartmentalization within the cell is critical for maintaining complex cellular processes and sustaining life. The sequestered environment in the nucleus is maintained by the nuclear pore complex (NPC), which controls the transport of proteins, nucleic acids, and other molecules between nucleus and cytoplasm. NPC is a cylindrical protein complex that fuses the internal and external nuclear membrane to form an aqueous channel (Cautain et al., 2015). The molecular mass is approximately 125,000 kDa with 100–150 nm in diameter (Lim et al., 2008). NPC consists of a central channel surrounded by three ring-like structures, the cytoplasmic ring, the central spoke ring, and the nuclear ring (Fig. 1). There are eight protein filaments on the cytoplasmic side and eight protein filaments on the nuclear side that are attached to this core structure. NPCs are built from approximately 30–50 proteins known as nucleoporins and are functionally conserved from yeast to mammals (Kau et al., 2004). These nucleoporins assemble in multiples of eight to generate 8-fold radial symmetry.

Molecules less than 40 kDa can passively move through the nuclear membrane. However, larger molecules require active transport and binding to the karyopherin-β family of proteins. These nuclear transport proteins are also known as importins and exportins. Table 1 lists the different human importins and exportins and their respective cargos. Importins bind to nuclear localizing sequences (NLS) on the cargo in the cytoplasm and are transported through the NPC into the nucleus (Fig. 2). Binding of RanGTP to the importins releases the cargo. The RanGTP–importin complex is then transported back to the cytoplasm where RanGTP is hydrolyzed to RanGDP by the GTPase-activating protein RanGAP. Importin is released and is available to bind to the next cargo. Conversely, for nuclear export, export complexes are formed consisting of exportins, RanGTP, and cargo with nuclear export signal (NES). The export signal is transported to the cytoplasm through the NPC due to the RanGTP:RanGDP gradient. RanGAP hydrolyzes RanGTP, leading to dissociation of the complex and release of cargo substrate in the cytoplasm. Exportins are transported back into the nucleus for recycling through NPC. RanGTP plays an important role in the nuclear-cytoplasm transport. Binding of RanGTP to importins in the cytoplasm helps in dissociation with cargo substrate, whereas binding to exportins in the nucleus leads to formation of the export complex. RanGTP increases the binding affinity of exportins by 500- to 1000-fold toward the cargo substrate. RanGTP is present in the nucleus at 100-fold greater concentration than in the cytoplasm due to Ran's guanine-exchange factor RCC1 (Bischoff & Ponstingl, 1995).

There are at least 20 known human karyopherin-β proteins that are involved in the nuclear transport of both importins and exportins (Tran et al., 2007). These proteins have similar molecular weights (95–145 kDa), acidic isoelectric points (4.0–5.5), low sequence identity (< 20%), and contain helical HEAT repeats (Chook & Suel, 2011). The N-terminal is responsible for binding Ran and is a conserved sequence. Importin-β is the most well characterized karyopherin-β proteins involved in transport of cargo from cytoplasm to the nucleus. Others include chromosome region maintenance 1 (CRM1), also known as exportin-1, and importin-β2. Importin-β is unique as it recognizes the NLS of the cargo with the help of adaptor protein importin-α. Other karyopherin-β proteins recognize the cargo substrate directly. Importin consists of 2 distinct domains: the N-terminal importin 1-binding domain and a C-terminal domain consisting of 10 tandem armadillo repeats. In the cytoplasm, the cargo bearing the NLS is recognized by importin-α initially, which in turn is associated with importin-β through N-terminal importin β-binding domain. This ternary complex is then actively transported through NPC into the nucleus. The importin β-binding domain on importin-α acts as a regulatory switch. When importin-α is not bound to cargo carrying NLS, the importin β-binding domain is not exposed to importin-β, preventing import of empty adaptor protein.

The classical NLS is basic amino acids that can be monopartite or bipartite (Lott & Cingolani, 2011). On the other hand, importin-β2 recognizes a more complex and diverse sequences called PY-NLS that consists of N-terminal basic motif and a C-terminal RX2-5PY motif. There are over 100 human proteins with PY-NLS that can potentially bind to importin-β2. All PY-NLSs bind at the same location, the concave surface of C-terminal arch on importin-β2. The PY-NLS binding region of importin-β2 remains relatively unchanged after binding to the different PY-NLSs.

There are at least 8 exportins that have been identified in eukaryotes. Most exportins such as CAS (cellular apoptosis susceptibility), exportin-t, and exportin-5 can transport a restricted number of cargo proteins (Guttler & Gorlich, 2011). These exportins have highly specialized function. CAS is involved in export of an NLS-free form of importin-α (Solsbacher et al., 1998). Exportin-t specializes in the export of tRNA and exportin-5 transports restricted number of cargo molecules including pre-micro RNA and double-stranded RNA (Kutay et al., 1998, Brownawell and Macara, 2002, Yi et al., 2003).

In contrast, exportin-1 can transport a wide variety of structurally unrelated cargoes (Stade et al., 1997). Exportin-1 is ubiquitous receptor protein that binds to leucine-rich sequences called NES. There are more than 200 macromolecules including proteins and RNA that are recognized by exportin-1. NES of the cargo protein binds to hydrophobic groove of exportin-1. Exportin-7 is another exportin with broad substrate specificity (Mingot et al., 2004).

In this review, we consider the fundamental aspects of nuclear transport from the perspectives of importins and exportins, and evidence that supports their role in cancer. We also discuss the importin and exportin drug targets currently in development and review the clinical experience. Finally, we present a detailed review of exportin-1 inhibitors, as these are the only specific drugs targeting importins and exportins that are currently being evaluated in human trials.