Survivin Isoforms Detection Predicts a Potential Role in Tumor Biology

Three main survivin isoforms demonstrated the ability to translocate to the surface of plasma membrane in multiple cell types, suggesting a targetable role as a molecular biomarker, according to findings presented at the AACR Virtual Annual Meeting II.¹

The 3 survivin detected were endogenous survivin (Isoforms 1), Δex3 and 2B on the surface of HEK293T embryonic kidney, HeLa cervical adenocarcinoma, U87 glioblastoma, and A1207 glioblastoma cells. Plasma membrane survivin expression was variable, however Δex3 showed robust membrane expression across all cell lines. Further, all 3 isoforms were determined to be secreted as exosomal cargo. By targeting these 3 isoforms, investigators may be able selectively and effectively destroy tumor cells.

“The 3 main survivin isoforms are all able to translocate to the surface plasma membrane of multiple cell types. Our findings support a role for the BIR domain, which is shared among the 3 isoforms, in transport to and/or retention on the plasma membrane,” the authors wrote in a poster presented as part of the virtual meeting.

Survivin, one of the key members of the inhibitor of apoptosis family of proteins, has been shown in the cytoplasm, mitochondria, nucleus, and the extracellular space. Its expression influences the tumor microenvironment and is thought to play an integral part in the biology of cancer, including progression, and response to therapy.^1,2

Structurally, survivin is composed of a 75 amino acid BIR (Baculoviral Inhibitor of apoptosis Repeat) domain linked to a 41 amino acid c-terminal alpha helix. Survivin functions as an adapter protein and interacts with various proteins within the cytoplasmic, nuclear, mitochondrial compartments. Multiple splice variants of surviving, including isoforms dEX3 and 2B, are known to play either pro- or anti-apoptotic roles and show various subcellular localizations.

Polymorphisms in the survivin gene are emerging as powerful tools to study the biology of the disease and have the potential to be used in disease prognosis and diagnosis.In this study, investigators sought to determine the mechanism for plasma membrane survivin translocation, as well as the localization of survivin isoforms in various cell lines.^1-3

Flow cytometry was used to measure relative surface expression through the staining of intact cells. The structural requirements for translocation were determined using mutant constructs.

Imaging flow cytometry analysis of cells using ImageStream, revealed polar localization of plasma membrane survivin, with specific punctate areas seen in 13% to 20% of cells. Secreted exosomes were examined for expression of survivin isoforms.

Investigators reported that in addition to canonical survivin (iso1), the ΔEX3 and 2B isoforms localized to the plasma membrane of several cell types. Endogenous survivin (isoform 1) is displayed at low levels on the surface of cancer cells and appears to localize to discrete, punctate areas of the membrane.

In a subsidiary analysis, deletion of a predicted transmembrane sequence in Δex3’s unique C-terminus—deletion of the first 10 amino acids—did not impair its translocation and ΔEX3 showed membrane translocation when overexpressed. This data demonstrated that treatment of cells with a protein transport reagent reduces membrane expression, implying that transport proceeds through typical ER/golgi pathways. Deletion of the first 10 amino acids shared by all 3 isoforms did impair plasma membrane localization of ΔEX3 and 2B in a cell type specific manner, however this did not affect isoform 1.

Heat shock stress applied to transfected 293T cells increased cell surface translocation and the secretion of all 3 isoforms in exosomes in addition to enriching the survivin protein in secreted exosomes.

Overexpression of survivin, an adapter protein that interacts with various proteins within the cytoplasmic, nuclear, mitochondrial compartments, has previously been reported in almost all human malignancies.^1,3 Survivin has been shown to be highly expressed in most cancers, is associated with a poor clinical outcomes, and has consistently been identified by molecular profiling analysis to be associated with high tumor grade cancers.^1,2 Results of the study support further investigation into the utility of survivin as a molecular target in manipulation tumor biology.

References

1. Figel S, Birkemeier M, Dharma S, Steinmetz E, Ciesielski M, Fenstermaker R. Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes. Poster presented at: American Association for Cancer Research Virtual Annual Meeting II. June 22-24, 2020. Abstract 2939.
2. Khan S, Ferguson Bennit H, Wall NR. The emerging role of exosomes in survivin secretion. Histol Histopathol. 2015;30(1):43-50. doi:10.14670/HH-30.43
3. Jaiswal PK, Goel A, Mittal RD. Survivin: a molecular biomarker in cancer. Indian J Med Res. 2015;141(4):389-397. doi:10.4103/0971-5916.159250