Kerry S. Wadey

Misregulation of the proline rich homeodomain (PRH/HHEX) protein in cancer cells and its consequences for tumour growth and invasion

The proline rich homeodomain protein (PRH), also known as haematopoietically expressed homeobox (HHEX), is an essential transcription factor in embryonic development and in the adult. The PRH protein forms oligomeric complexes that bind to tandemly repeated PRH recognition sequences within or at a distance from PRH-target genes and recruit a variety of PRH-interacting proteins. PRH can also bind to other transcription factors and co-regulate specific target genes either directly through DNA binding, or indirectly through effects on the activity of its partner proteins. In addition, like some other homeodomain proteins, PRH can regulate the translation of specific mRNAs. Altered PRH expression and altered PRH intracellular localisation, are associated with breast cancer, liver cancer and thyroid cancer and some subtypes of leukaemia. This is consistent with the involvement of multiple PRH-interacting proteins, including the oncoprotein c-Myc, translation initiation factor 4E (eIF4E), and the promyelocytic leukaemia protein (PML), in the control of cell proliferation and cell survival. Similarly, multiple PRH target genes, including the genes encoding vascular endothelial growth factor (VEGF), VEGF receptors, Endoglin, and Goosecoid, are known to be important in the control of cell proliferation and cell survival and/or the regulation of cell migration and invasion. In this review, we summarise the evidence that implicates PRH in tumourigenesis and we review the data that suggests PRH levels could be useful in cancer prognosis and in the choice of treatment options.

Soluble N-cadherin: A novel inhibitor of VSMC proliferation and intimal thickening

Reoccurrence of symptoms occurs in 30–50% of coronary artery disease patients receiving vein grafts or bare-metal stents due to intimal thickening (restenosis). Restenosis is caused by vascular smooth muscle cell (VSMC) migration and proliferation. New therapeutic approaches that reduce VSMC migration and proliferation while promoting endothelial cell (EC) coverage are required. We assessed the effect of a soluble form of N-cadherin (SNC-Fc, a fusion of the extracellular portion of N-Cadherin to a mutated Fc fragment of IgG), a cell–cell junction molecule, on human saphenous VSMC proliferation and migration in vitro. We also assessed its effect on intimal thickening in a validated human ex vivo organ culture model. We observed that SNC-Fc significantly inhibited VSMC proliferation and to a lesser extent migration. The anti-proliferative effect of SNC-Fc was mediated by the interaction of SNC-Fc with the FGFR, rather than through inhibition of β-catenin signalling. SNC-Fc also significantly reduced intimal thickening by ~ 85% in the ex vivo organ culture model. SNC-Fc treatment inhibited proliferation of the intimal cells but did not affect migration. SNC-Fc reduced EC apoptosis, without detrimental effects on EC proliferation and migration in vitro. Importantly SNC-Fc increased EC coverage in the ex vivo model of intimal thickening. In conclusion, we suggest that SNC-Fc may have potential as an anti-proliferative therapeutic agent for reducing restenosis which has no detrimental effects on endothelial cells.

Protein kinase CK2 inhibition suppresses neointima formation via a proline-rich homeodomain-dependent mechanism

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser163 and Ser177 within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells.

Targeting Wnt/β-Catenin Activated Cells with Dominant-Negative N-cadherin to Reduce Neointima Formation

Approximately 50% of coronary artery bypass grafts using the autologous saphenous vein fail within 10 years due to intimal thickening. This study examined whether a gene therapy approach that selectively kills Wnt/β-catenin/T cell factor (TCF) activated vascular smooth muscle cells (VSMCs) using dominant-negative N-cadherin (dn-N-cadherin) reduced intimal thickening. Cultured human VSMCs infected with an adenovirus (Ad) encoding dn-N-cadherin via the TCF promoter (Ad-TOP-dn-N-cadherin) specifically expressed dn-N-cadherin in response to activation of the Wnt/β-catenin/TCF pathway. Infection with Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis (3 ± 0.2% versus 9 ± 0.7%; p < 0.05, n = 6) and significantly inhibited VSMC migration by 83 ± 15% (p < 0.05, n = 6), but did not affect VSMC proliferation (p > 0.05, n = 5). In an ex vivo human saphenous vein organ culture model, luminal delivery of Ad-TOP-dn-N-cadherin significantly increased VSMC apoptosis after 7 days of culture (4 ± 1.4% versus 9 ± 1.6%; p < 0.01, n = 6) and suppressed intimal thickening by 75 ± 7% (p < 0.05, n = 5), without a detrimental effect on endothelial cell coverage. In vivo, Ad-TOP-dn-N-cadherin significantly reduced intimal thickening at day 21 (n = 10) in comparison to the Ad-β-galactosidase (Ad-β-gal) control virus (n = 12, p < 0.05) in the mouse carotid artery ligation model. In summary, we have developed a novel approach to selectively reduce intimal thickening, which may be beneficial in reducing late vein graft failure.

Aneurysm Severity is Increased by Combined Mmp-7 Deletion and N-cadherin Mimetic (EC4-Fc) Over-Expression

There is an unmet need for treatments to reduce abdominal aortic aneurysm (AAA) progression. Vascular smooth muscle cell (VSMC) apoptosis precipitates AAA formation, whereas VSMC proliferation repairs the vessel wall. We previously demonstrated that over-expression of EC4-Fc (truncated N-cadherin), or deletion of matrix-metalloproteinase-7 (Mmp-7) reduced VSMC apoptosis in mouse atherosclerotic plaques. Additionally, MMP-7 promotes VSMC apoptosis by cleavage of N-cadherin. We investigated their combined effect on AAA formation. Increased apoptosis and proliferation were observed in human AAA (HAAA) sections compared to normal aortae (HA). This coincided with increased MMP-7 activity and reduced N-cadherin protein levels in HAAA sections compared to HA. Using a mouse model of aneurysm formation, we showed that the combination of Mmp-7 deletion and EC4-Fc overexpression significantly increased AAA severity. Medial apoptosis and proliferation were both significantly reduced in these mice compared to control mice. In vitro, MMP-7 inhibition and EC4-Fc administration significantly supressed human aortic VSMC apoptosis (via activation of PI-3 kinase/Akt signalling) and proliferation. In conclusion, combined Mmp-7 deletion and systemic over-expression of EC4-Fc reduced both proliferation and apoptosis. Reduced proliferation-mediated repair over-rides any benefit of reduced apoptosis, increasing aneurysm severity. Future studies should therefore focus on retarding VSMC apoptosis whilst promoting VSMC proliferation.