Therapeutic Inhibition of XPO1-Mediated Nucleocytoplasmic Transport With SINE Technology

Cancer is a complex disease characterized by uncontrolled cell growth and metastatic dissemination of tumor cells. Six complementary capabilities and tumorigenic properties that are acquired during tumorigenesis and represent the hallmarks of cancer are sustained proliferative signaling, evasion of growth suppressors, resistance to cell death/apoptosis, enabling of replicative immortality, induction of angiogenesis, and activation of invasion and metastasis.¹ These essential features define a multistep evolutionary process whereby normal cells progressively become cancerous, acquiring the capacity to proliferate, survive, invade, and disseminate.¹

Cancer cells acquire these properties through different mechanisms; for example, the patterns of genetic mutations appear to differ between cancer types, although genomic instability is an omnipresent enabling characteristic in carcinogenesis.¹ In addition to these core hallmarks, other characteristics, including metabolic reprogramming or dysregulation of cellular energetics and avoidance of immune destruction, have been identified as emerging hallmarks in cancer pathogenesis.¹

The hallmark features of cancer are coordinated by molecular circuits which relay signals from the microenvironment of the incipient cancer to and from the nucleus, the protected housing for the genome and the hub of gene expression programs. The dynamic nucleocytoplasmic shuttling of key regulatory proteins, including tumor suppressors, cell-cycle regulators, and master transcription factors, is an essential part of this tumor-microenvironment relay system.² Tumor suppressors with critical functions in cell-cycle regulation, apoptosis, and DNA damage response and repair, including p53, forkhead box O1 (FOXO1), and adenomatous polyposis coli (APC), undergo nucleocytoplasmic shuttling and have dysregulated function in many cancers.^2,3

The function of tumor suppressors is closely associated with their location; transcription factors, such as p53, reside in the nucleus, whereas other proteins, such as the DNA repair-associated BRCA1, can be found in the nucleus and cytoplasm of normal cells. In cancer, p53 can become mislocalized to the cytoplasm, whereas BRCA1 may be mislocalized to the nucleus.^2,4 The aberrant nuclear exclusion of p53 has been noted in many cancers, including neuroblastomas, retinoblastomas, and ovarian and colorectal cancers.² Mutations in p53 are widespread and one of the most common in cancer; however, the inactivation of wild-type p53 via abnormal cytoplasmic localization, in effect abrogates p53 function. This mode of p53 inactivation may be reversible and would require strategies for returning the intact functional wild-type p53 protein to the nucleus, its proper home. Indeed, studies in neuroblastoma cell lines showed that restoration of p53 to the nucleus also restores its tumor suppressor function.² A study that utilized small-molecule inhibitors of nuclear export protein exportin 1 (XPO1), also called chromosome region maintenance 1 (CRM1), demonstrated that XPO1 induces nuclear accumulation of p53 and phosphorylated mitogen-activated protein kinase (pMAPK), which, in turn, promoted apoptosis in human melanoma cells.⁵

The cyclin-dependent kinase (CDK) inhibitor p27 is an example of another class of nuclear proteins that is aberrantly localized in cancers.4 Aberrant phosphorylation of p27 at serine 10, due to constitutive Akt activation, leads to its mislocalization to the cytoplasm. In the clinical setting, the presence of elevated levels of cytoplasmic serine 10-phosphorylated p27 has been correlated with the grade and prognosis of gliomas.⁴ Unlike p53, p27 is rarely mutated in cancers, but it can be aberrantly transported to the cytoplasm, where it may function as an oncogene to drive cell migration.³

Inhibition of nuclear export can restore p27 to the nucleus in tumor cells, serving as another example of the potential for using nuclear export/import systems as therapeutic targets in cancer.³

Forkhead box O (FOXO) proteins are a family of transcription factors; their activity is regulated via subcellular localization, DNA binding, and degradation.⁴ All FOXO proteins contain nuclear localization signals (NLSs), and phosphorylation of FOXO by Akt interferes with NLS function, thereby impeding nuclear transport of FOXO.⁴ FOXO proteins also contain a nuclear export signal (NES), which mediates XPO1 binding to non-DNA bound nuclear FOXO, enabling its export from the nucleus.⁴ Based on the observation that apoptosis is induced upon forced expression of nuclear FOXO in cancer cells, restoration of wild-type FOXO to the nucleus by manipulation of the nucleocytoplasmic shuttling machinery has been proposed as a promising therapeutic strategy in cancer.⁴