Effect of MRE11 loss on PARP-inhibitor sensitivity in endometrial cancer in vitro.

AIM OF THE STUDY: To evaluate the frequency of MRE11/RAD50/NBS1 (MRN)-complex loss of protein expression in endometrial cancers (EC) and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose) polymerase (PARP)-inhibitory treatment. METHODS: MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T) allele in MRE11 was sequenced in selected cases (n = 26). Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR) DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment. RESULTS: Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T) mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells. CONCLUSION: Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.

The carboxyl terminus of the Galpha-subunit is the latch for triggered activation of heterotrimeric G proteins.

The receptor-mimetic peptide D2N, derived from the cytoplasmic domain of the D(2) dopamine receptor, activates G protein alpha-subunits (G(i) and G(o)) directly. Using D2N, we tested the current hypotheses on the mechanism of receptor-mediated G protein activation, which differ by the role assigned to the Gbetagamma-subunit: 1) a receptor-prompted movement of Gbetagamma is needed to open up the nucleotide exit pathway ("gear-shift" and "lever-arm" model) or 2) the receptor first engages Gbetagamma and then triggers GDP release by interacting with the carboxyl (C) terminus of Galpha (the "sequential-fit" model). Our results with D2N were compatible with the latter hypothesis. D2N bound to the extreme C terminus of the alpha-subunit and caused a conformational change that was transmitted to the switch regions. Hence, D2N led to a decline in the intrinsic tryptophan fluorescence, increased the guanine nucleotide exchange rate, and modulated the Mg(2+) control of nucleotide binding. A structural alteration in the outer portion of helix alpha5 (substitution of an isoleucine by proline) blunted the stimulatory action of D2N. This confirms that helix alpha5 links the guanine nucleotide binding pocket to the receptor contact site on the G protein. However, neither the alpha-subunit amino terminus (as a lever-arm) nor Gbetagamma was required for D2N-mediated activation; conversely, assembly of the Galphabetagamma heterotrimer stabilized the GDP-bound species and required an increased D2N concentration for activation. We propose that the receptor can engage the C terminus of the alpha-subunit to destabilize nucleotide binding from the "back side" of the nucleotide binding pocket.