by ecancer reporter Clare Sampson

Inhibitors of the enzyme poly (ADP-ribose) polymerase (PARP) are showing promise as treatments for several types of cancer and one PARP inhibitor, olaparib, has been approved by the FDA for advanced ovarian cancer in patients with mutations in the BRCA genes.

This enzyme catalyses the addition of one or more ADP-ribose units to its target proteins, which include histones.

Addition of ADP-ribose to these proteins destabilises the structure of chromatin, thus allowing other proteins, including DNA repair proteins, to access chromosomal DNA.

Inhibiting this activity is therefore, in theory, likely to prevent the repair of DNA damage and thus promote the death of tumour cells.

This function is particularly effective in tumour cells that lack functioning copies of the proteins that are encoded by the BRCA genes, which are also involved in DNA repair.

Olaparib has also been approved to treat triple negative breast cancer (TNBC) in patients with BRCA mutations, but reported clinical results have been mixed with many tumours showing resistance to the drug.

A group of researchers led by Mien-Chie Hung at the University of Texas MD Anderson Cancer Center, Houston, Texas, USA therefore set out to investigate the molecular mechanisms of resistance to PARP inhibitors in this form of breast cancer.

They first observed that TNBC cells show more oxidative DNA damage than cells from other types of breast cancer: this type of DNA damage, which is caused by reactive oxygen species (ROS), stimulates PARP1 activity.

Reactive oxygen species also activate receptor tyrosine kinases, which are often over-expressed in TNBC cells.

Hung and his co-workers investigated the interaction between PARP and these kinases, firstly searching for specific kinases that associate with PARP under oxidative stress.

They identified three such kinase genes: ERBB3, FLT3, and HGFR, which encodes the c-Met protein.

High c-Met expression levels are known to correlate with poor survival in patients with TNBC.

The researchers showed that a fraction of the c-Met protein is translocated into the nucleus, where it interacts with the nuclear protein PARP1.
This interaction requires c-Met to be active as a kinase and is enhanced by treatment with the reactive oxygen species hydrogen peroxide (H2O2).

This, and other results, suggested that transport of c-Met into the nucleus and its interaction with PARP1 is induced by oxidative stress.

Knockdown of c-Met in a TNBC cell line using a small hairpin RNA sensitised the tumour cells to inhibition by olaparib and by two PARP inhibitors in clinical trials, veliparib and rucaparib, and similar results were obtained using small-molecule c-Met inhibitors.

Hung and his colleagues speculated that differences in c-Met expression levels might be responsible for the differential response of untreated BRCA negative cell lines to PARP inhibitors.

They tested this by knocking down BRCA expression levels in breast cancer cell lines with wild type BRCA and exposing those cells to PARP inhibitors, and found that only those cell lines with low c-Met levels became sensitive to PARP inhibition.

Taken together, these results suggest that increased expression of c-Met induces PARP resistance in breast cancer cells.

Furthermore, cells with knocked down c-Met expression showed higher levels of oxidative DNA damage than control cells, thus providing a mechanism for sensitivity to further DNA damage induced by a PARP inhibitor.

The researchers then set out to discover whether the interaction between PARP1 and c-Met under oxidative stress involved phosphorylation of PARP1 by the kinase.

Bioinformatics analysis predicted a tyrosine residue in the catalytic domain of PARP1, tyrosine 907 (Y907) as a likely site of c-Met phosphorylation.

Hung and his co-workers expressed wild type PARP1 and two Y907 mutants – Y907F, which cannot be phosphorylated, and Y907E, in which the negative charge of the glutamic acid can mimic a phosphate group, in TNBC cells without PARP1 – and measured oxidative DNA damage.

Cells expressing wild type PARP1 and the ‘phosphomimetic’ Y907E mutant showed more poly (ADP-ribose) polymerase activity but less oxidative DNA damage than those expressing Y907F.

PARP enzymes that were phosphorylated at this position were also shown to be less sensitive to anti-tumour PARP inhibitors than the unphosphorylated enzymes. 

Knocking down c-Met expression, and therefore Y907 phosphorylation, re-sensitised cells bearing wild type but not Y907E PARP to the PARP inhibitors.

Finally, the researchers showed that a combination of PARP inhibitors with c-Met inhibitors suppressed the growth of tumour cells in several human TNBC and mouse mammary tumour cell lines, and in mouse xenograft models of breast and lung cancer

These results suggest that a combination of c-Met and PARP inhibitors may be an effective therapy for patients with BRCA negative tumours that do not respond to PARP inhibition alone.

Reference: Du, Y., Yamaguchi, H., Wei, Y. and 22 others (2016). Blocking c-Met–mediated PARP1 phosphorylation enhances anti-tumor effects of PARP inhibitors. Nature Medicine, published online ahead of print 18 January 2016. doi:10.1038/nm.4032

Source: Nature Medicine