EPLIN: a fundamental actin regulator in cancer metastasis?
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Treatment of malignant disease is of paramount importance in modern medicine. In 2012, it was estimated that 162,000 people died from cancer in the UK which illustrates a fundamental problem. Traditional treatments for cancer have various drawbacks, and this creates a considerable need for specific, molecular targets to overcome cancer spread. Epithelial protein lost in neoplasm (EPLIN) is an actin-associated molecule which has been implicated in the development and progression of various cancers including breast, prostate, oesophageal and lung where EPLIN expression is frequently lost as the cancer progresses. EPLIN is important in the regulation of actin dynamics and has multiple associations at epithelial cells junctions. Thus, EPLIN loss in cancer may have significant effects on cancer cell migration and invasion, increasing metastatic potential. Overexpression of EPLIN has proved to be an effective tool for manipulating cancerous traits such as reducing cell growth and cell motility and rendering cells less invasive illustrating the therapeutic potential of EPLIN. Here, we review the current state of knowledge of EPLIN, highlighting EPLIN involvement in regulating cytoskeletal dynamics, signalling pathways and implications in cancer and metastasis.
KeywordsEPLIN Cancer Metastasis Actin
The incidence of cancer is slowly rising and has become a global burden. A fundamental reason why cancer is such a problem is because of its ability to spread, invade surrounding tissue and potentially form secondary cancers at distinct sites around the body by metastasis. Cancer hallmarks include uncontrolled cell growth and evasion of cell death, and this ultimately can lead to tumour formation. According to the World Health Organisation (WHO), 8.2 million people died from cancer in 2012 worldwide . In the UK alone, mortality rates reached 162,000 annual deaths . This illustrates a considerable need for better treatment, diagnosis and management of the disease. Epithelial protein lost in neoplasm (EPLIN) is a molecule involved in regulation of the actin cytoskeleton and has been implicated in the development and progression of various cancer types, displaying frequent downregulation or loss in cancer, creating a potential for prognostic targeting and as a tumour suppressor. This current review discusses EPLIN’s role in actin dynamics and in the pathophysiology of cancer development and progression.
2 Epithelial protein lost in neoplasm
2.1 The epithelial protein lost in neoplasm interactome: regulation in actin dynamics
Biological significance and reference
Actin is an abundant protein important for cell migration. EPLIN has two actin-binding domains that flank the EPLIN LIM domain. Pull-down assays revealed that EPLIN binds actin monomers, and this results in actin cross linking and actin filament bundle assembly.
May form a complex with EPLIN to co-ordinate actin dynamics. IHC of PCa tissue vs normal reveals that EPLIN overexpression influences paxillin expression and localisation. Co-localisation, co-precipitation and an in situ proximal ligation assay revealed direct association between the two molecules in cultured human mesangial cells.
Immunoprecipitation and GST pull-down assays reveal that EPLIN interacts with α-catenin, forming a cadherin–β-catenin–α-catenin–EPLIN complex.
In vivo co-localisation studies and in vitro GST pull-down assays reveal that EPLIN interacts with the peripheral membrane protein, supervillain.
Pull-down assays reveal that endogenous EPLIN co-immunoprecipitates with endogenous PINCH-1 in keratinocytes.
ERK phosphorylates EPLIN and decreases EPLIN affinity to F-actin promoting cell migration. Inhibition of ERK abolishes EPLIN expression and reduces tumour-suppressive ability of EPLIN.
In melanoma cells, both EPLIN isoforms are inhibited by DNp73, and this drives a more invasive phenotype.
EPLIN is differentially regulated by the DNA-binding protein, SATB2. SATB2 regulates the actin cytoskeleton via EPLIN association. When SATB2 is knocked out, osteosarcoma cells show reduced migration and are less invasive, and this is mediated by EPLIN.
EPLIN regulates the lipid raft tumour-suppressive protein, Cav-1. Co-immunoprecipitation and mass spectroscopy analysis revealed that EPLIN and Cav-1 bind to each other in normal and RasV12 cells.
2.2 The adherens junction
2.3 Epithelial protein lost in neoplasm—a key player in cell division?
Cell division is the splitting of one cell into two, where biological information is passed onto daughter cells. For this process to successfully occur, various proteins need to functionally regulate the division and these include Rho GTPases, cyclin-dependant kinases, integrins, cdc42, focal adhesion kinases, myosin and the globular protein actin . With this in mind, an actin-binding protein like EPLIN may potentially have a regulatory role in cell division. This has been recently shown using HeLa cells where EPLIN depletion resulted in large numbers of multinucleated cells, signifying cytokinesis failure during cell division . In successful mitotic division, actin and myosin II accumulate at the cleavage furrow during cytokinesis and EPLIN loss compromised each protein’s ability to efficiently do this . EPLIN appears to be important for the accumulation of other mitotic regulatory proteins including the GTPases RhoA and cdc42, where EPLIN depletion resulted in either a significantly reduced concentration of RhoA or a misplaced location of cdc42 at the cleavage furrow . EPLIN aids this successful cell division in conjunction with a number of regulatory proteins including supervillin and the oncogenic kinesin, KIF14, suggesting a complex network of regulatory proteins at the cleavage furrow . Altogether, this suggests that EPLIN may have an integral role in cytokinesis and loss may lead to aneuploidy and genomic instability of daughter cells . Therefore, EPLIN is crucial to co-ordinate actin and myosin dynamics throughout cell division and loss in cancer cells could have downstream effects on successful cytokinesis, increasing their tendency to form a cancer .
2.4 Post-translational modification
3 The role of epithelial protein lost in neoplasm in cancer
Cancer progression involves various cellular, morphological and molecular alterations which result in a transformed cellular phenotype, ultimately having the potential to invade surrounding tissue and disseminate throughout the body. Cancer treatment options remain largely unspecific and create various undesired side effects. Therefore, elucidating a molecular target for treating cancer, in addition to understanding the mechanism of cancer development, is crucial. EPLIN first received attention for its involvement in cancer in 1999 where EPLIN downregulation was described in various cancer cell lines . Altogether, low levels of EPLIN transcript were found in 8/8 oral cancer cell lines, 5/6 breast cancer cell lines and 4/4 prostate cancer cell lines . Using PC-3 and DU-145 prostate cancer cell lines, EPLIN expression was significantly reduced compared to primary prostate epithelial cells (PrEC), whereas the prostate specific antigen (PSA) positive LNCaP and LAPC4 prostate cancer cell lines failed to express EPLINα at all . This notion of EPLIN loss is also seen in breast cancer where EPLIN expression in cell lines BT-20, SKBr-3, MCF-7, T-47D and MDA-MB-231 was either reduced or completely lost . Lastly, the authors demonstrated EPLIN as a putative tumour suppressor molecule, where overexpression of EPLINα caused a reduction in cancer cell growth . Interestingly, when EPLINα was depleted in breast cancer cell lines, EPLINβ either remained consistent or actually increased . This illustrates the potential cancer protective effects that the EPLINα isoform may exert in various cancer cell systems. EPLIN overexpression has also proved effective in altering the growth phenotype and morphology in additional cell systems including anchorage-independent NIH3T3 transformed cells . Using a soft agar assay and utilising the activated Cdc42 or the chimeric nuclear oncogene EWS/Fli-1 to transform NIH3T3 cells, EPLIN overexpression resulted in a ∼80 % decrease in colony formation for Cdc42 transformed cells, with a similar growth decrease in EWS/Fli-1 transformed cells . Interestingly, EPLIN displayed heterogeneous staining throughout Ras cells rather than localisation to the actin cytoskeleton . This implies that oncogenic transformation affects the EPLIN/actin architecture, and thus, the localisation of EPLIN to the actin cytoskeleton may be important to exert its suppressive ability .
3.1 Prostate cancer
3.2 Breast cancer
Our lab has recently evaluated EPLIN involvement in cancer progression in a number of model systems [10, 11, 12, 13, 14]. By comparing EPLINα IHC staining in normal vs tumour cells in breast cancer progression, EPLINα was found to be substantially weaker in tumour cells than in normal epithelial cells (see Fig. 7b) . This correlated with lower EPLINα transcript in tumour samples compared to normal samples (see Fig. 7e) with lower EPLIN levels being associated with higher tumour grade, a poorer patient prognosis and reduced overall survival rates (see Fig. 7f–h) . IHC analyses in breast cancer from additional research groups also show EPLIN loss as the tumour becomes more aggressive, specifically comparing EPLIN immunointensity of primary tumours vs tumours with lymph node metastases . Lastly, in vitro and in vivo overexpression analysis of EPLIN highlighted significant reductions in cell growth and cell invasion using transfected breast cancer cell lines, and also, highly significant reductions in tumour size were observed in nude mice inoculated with EPLIN-α-transfected vs control MDA-MB-231 breast cancer cells .
3.3 Further pathological implications
Summary of EPLIN clinical implications
(1) Immunoblot analyses demonstrated that EPLIN expression in prostate cancer cell lines and xenograft tumours is reduced compared to prostate epithelial cells (PcEC). PC-3, DU-145, LNCaP, LAPC4, LAPC3 and LAPC9 all displayed loss of EPLIN protein.
(2) IHC analyses of normal and cancerous clinical prostate sections revealed a greatly reduced staining pattern of EPLIN in tumour samples. Overexpression of EPLIN in PC-3 cells negatively impacted cell growth in vitro and in vivo, and were less invasive and had reduced adhesion to the ECM.
(3) EPLIN is implicated in the process of EMT, and IHC analysis revealed that EPLIN loss is correlated with prostate cancer progression, with a significant reduction of EPLIN expression in tissues with lymph node metastases compared to primary tumours and normal prostate tissues.
(1) Immunoblot analysis revealed reduced or abolished expression of EPLIN protein in tumourigenic breast cancer cell lines (BT-20, SK-Br-3, MCF-7, T-47D and MDA-MB-231) compared to mammary epithelial cells (MEC), immortalised mammary epithelial cells (IMEC) and a non-tumourigenic breast cancer cell line, HBL-100.
(2) Analysis by Q-PCR revealed lower levels of EPLINα in tumour samples compared to normal. Higher-tumour-grade samples had lower EPLIN transcript. Patients with poorer prognosis and patients who died of the cancer had significantly lower levels of EPLIN transcript. Overexpression of EPLIN rendered cells less invasive, and had a reduced growth rate in vitro and in vivo and were less motile.
(3) IHC analyses displayed a reduction in EPLIN staining in tissues of breast cancer lymph node metastases compared to primary breast tumours.
Q-PCR analyses revealed lower levels of EPLINα transcript in tumour tissues compared to normal. Higher-tumour-grade samples had lower EPLIN transcript. Patients who died of the cancer had significantly lower levels of EPLIN transcript. Patients with local advanced T stage cancer (T2–T4) and patients with lymphatic metastasis had lower levels of EPLINα transcript. Overexpression analysis caused cells to be less invasive and to have a reduced growth rate in vitro and in vivo.
Q-PCR analyses revealed reduced levels of EPLINα transcript in tumour samples compared to normal. Tissues of a higher TNM stage and where there was nodal involvement also had lower EPLIN transcript. Overexpression analysis revealed a reduction of cell growth and motility in the SKMES-1 cell line.
IHC analyses revealed that EPLIN is significantly reduced in lymph node metastatic tumours compared to primary tumours in colorectal cancer.
IHC analyses revealed a reduction of EPLIN staining of cancerous tissue with lymph node metastasis compared to primary tumours.
Northern analyses determined that EPLIN expression in 8/8 oral cancer cell lines is reduced compared to control G3PDH.
Collectively, these studies suggest EPLIN may be a clinical indicator for cancer progression in addition to providing further evidence of a tumour-suppressive role for EPLINα in the regulation of cancer progression.
Finally, in addition to the implication of EPLIN in the spread and progression of cancer, a recent publication provides a link between EPLIN and renal diseases where patients with either membranoproliferative glomerulonephritis (MPGN) or IgA nephropathy had a decreased expression profile for EPLIN via IHC analysis . This advocates the idea that EPLIN may be involved in the pathology of various disease states.
Angiogenesis is the formation of new blood vessels from pre-existing vessels and is essential for wound healing and normal growth and development. The angiogenic process is frequently utilised by cancer cells, by a means of metastasis, to reach secondary sites around the body and develop secondary tumours. Angiogenesis is therefore a critical factor when targeting cancer therapies. EPLINα demonstrates a suppressive role in angiogenesis, where overexpression analysis in the HECV endothelial cell line resulted in a reduced capacity to generate tubular structures in a Matrigel tubule formation assay when compared to vector controls . This regulatory effect was also apparent in vivo where mice injected with HECV cells overexpressing EPLINα in conjunction with cancer cells developed tumours significantly slower than controls . Forced expression also appears to exert an effect on cell matrix adhesion and migration capabilities in this cell line where cells overexpressing EPLINα both migrated at a significantly slower rate and were significantly less able to adhere to the Matrigel basement membrane . This suppressive role in angiogenesis illustrates that EPLINα has potentially various regulatory mechanisms for reducing cancer metastasis and could be an effective target for cancer therapy.
4 Conclusions and outlook
The authors are grateful to Cancer Research Wales and the Welsh Life Science Network - Ser Cymru for supporting this work.
Conflict of interest
The authors declare that they have no conflict of interest.
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