by ecancer reporter Clare Sansom

Targeted cancer therapy offers significant advantages over chemotherapy, but it can be ineffective in treating some aggressive tumours because pathways that are blocked can be bypassed.

Few, if any, anti-cancer agents that block multiple oncogenic pathways are yet available.

Phosphorylation of serine and threonine residues that occur immediately before proline in amino acid sequences (proline-directed phosphorylation) is a key signalling mechanism in several oncogenic pathways.

A proline isomerase enzyme, Pin1, coordinates several of these pathways through catalysing the cis/trans isomerisation of proline residues that follow phosphorylated serine and threonine in both oncogenes and tumour suppressor genes.

Pin1 is commonly activated or over-expressed in human cancers, and this has been associated with poor prognosis; in contrast, polymorphisms that lower Pin1 expression are associated with a reduction in cancer risk.

Pin1 can therefore be considered a valid target for a drug to block multiple oncogenic pathways, but no potent and specific inhibitors of this enzyme are yet known.

A team of researchers led by Xiao Zhen Zhou and Kun Ping Lu at Harvard Medical School, Boston, Massachusetts, USA designed a mechanism-based high throughput screen to identify inhibitors of this enzyme.

This screen used fluorescence polarisation to select compounds that could compete with the peptide inhibitor pTide for binding to the Pin1 active site.

About 8,200 compounds were tested in this screen, and the highest-binding compound was identified as 13-cis-retinioic acid.

An isomer of this compound, all-trans retinoic acid (ATRA) initially bound to Pin1 more tightly still, but this difference disappeared after longer incubation times; this suggested that the 13-cis compound might bind to the active site after conversion to the trans form.

Further experiments confirmed that ATRA was a sub-micromolar inhibitor of Pin1, with a Ki value of 0.82 μM.

The researchers suggested that the carboxylic acid group in ATRA could act as an alternative to the phosphate of Pin1 substrates in binding to the enzyme’s active site, and tested this by screening structurally similar retinoids without this group against the enzyme.

No retinoid without a carboxylate group showed any inhibitory activity against Pin1.

Zhou, Lu and their colleagues then determined the structure of the isomerase domain of Pin1 with ATRA bound.

The carboxylate group of the inhibitor was clearly seen in the active site, making salt bridges with two positively charged amino acids that are critical for the enzyme’s activity, K63 and R69.

ATRA was also found to induce the degradation of Pin1, and to inhibit several oncogenic phenotypes that have been associated with the over-expression of this protein in mouse embryonic fibroblast (MEF) cells.

This compound has already been approved as a treatment for acute promyelocytic leukaemia (APL) through a mechanism involving the degradation of the fusion onco-protein promyelocytic leukaemia–retinoic acid receptor α (PML–RAR-α).

Degradation of PML–RAR-α has been associated with the phosphorylation of a Ser-Pro motif that corresponds to a known Pin1 binding site in the receptor.

The researchers showed that Pin1 inhibition with ATRA was sufficient to degrade this fusion protein in APL cells and inhibit cell growth.

Microarray analysis showed similarities in gene expression patterns between APL cells treated with ATRA and cells in which Pin1 expression had been knocked down with a small hairpin RNA.

ATRA, but not some less potent Pin1 inhibitors, was shown to induce differentiation in APL cells and reduce Pin1 levels in bone marrow in a mouse model of this type of leukaemia, and ATRA was also less toxic than the other inhibitors.

Similarly, cells from human patients with APL were found to contain high levels of both Pin1 and PML–RAR-α; these were reduced after treatment with ATRA and, interestingly, in cells from individuals in complete APL remission.

Finally, the researchers tested the hypothesis that ATRA might be active against other Pin1-dependent cancer types using several different breast cancer cell lines.

The inhibitor was found to reduce the abundance of a number of onco-proteins that are known to be Pin1 substrates, as well as of Pin1 itself, and to inhibit the growth of human triple-negative breast cancer cells in vitro.

Taken together, these results suggest that ATRA, a potent inhibitor of Pin1, can inhibit a number of oncogenic pathways that depend on this enzyme and may be a useful drug in APL, triple negative breast cancer and perhaps other aggressive tumour types.

Reference

Shuo Wei, S., Kozono, S., Kats, L. et al.,  Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer. Nature Medicine, published online ahead of print 13 April 2015.