Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

A recently discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The non-enzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could non-physiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1Δppn2Δ, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain

Reciprocal regulation between GCN2 (eIF2AK4) and PERK (eIF2AK3) through the JNK-FOXO3 axis to modulate cancer drug resistance and clonal survival

Pharmaceutical inhibitors of the endoplasmic reticulum (ER)-stress modulator PERK (eIF2AK3) have demonstrated anticancer activities in combination therapies, but their effectiveness as a single agent is limited, suggesting the existence of possible compensatory cellular responses. To explore the potential mechanisms involved, we performed time-course drug treatment experiments on the parental MCF-7 and drug resistant MCF-7EpiR and MCF-7TaxR breast cancer cells and identified GCN2 (eIF2AK4) as a molecule that can potentially cooperate with PERK to regulate FOXO3 via JNK and AKT to modulate drug response. Consistently, GCN2 knockdown severely impaired the clonal survival of parental and resistant MCF-7 cells and sensitised them to epirubicin and paclitaxel treatment. Western blot, RT-qPCR and ChIP analyses also confirmed that GCN2 inactivation causes an induction of JNK and thereby FOXO3 activity, culminating in an increase in PERK activity and expression at the transcription level. Conversely, PERK-inactivation using GSK2606414-induces an induction in GCN2 expression and activity also associated with JNK. In agreement, we also showed that the perk −/− MEFs, expressing elevated levels of P-JNK, JNK, GCN2 and reduced levels of P-AKT and P-FOXO3, have lower clonogenicity and are more sensitive to epirubicin compared to wild-type MEFs. Similarly, gcn2 −/− MEFs expressing augmented levels of P-JNK, JNK, P-PERK, PERK and lower levels of P-AKT and P-FOXO3 also had lower clonogenicity and were more sensitive to epirubicin and PERK-inhibition. In addition, JNK1/2 deletion in MEFs resulted in reduced levels of GCN2, FOXO3, PERK, P-PERK expression as well as FOXO3 activity and enhanced clonal survival and resistance to PERK-inhibition. Together these results demonstrate that GCN2 cooperates with PERK through the JNK-FOXO3 axis in a reciprocal negative feedback loop to mediate cancer chemotherapeutic drug response and clonal survival, advocating the potential of targeting GCN2 as a therapeutic strategy for treating cancer and for overcoming drug resistance

FOXM1 modulates 5-fluorouracil sensitivity in cholangiocarcinoma through Thymidylate synthase (TYMS) : implications of FOXM1-TYMS axis uncoupling in 5-FU resistance

Fluorouracil (5-FU) is thefirst-line chemotherapeutic drug for cholangiocarcinoma (CCA), but its efficacy has beencompromised by the development of resistance. Development of 5-FU resistance is associated with elevatedexpression of its cellular target, thymidylate synthase (TYMS). E2F1 transcription factor has previously been shown tomodulate the expression of FOXM1 and TYMS. Immunohistochemical (IHC) analysis revealed a strong correlatedupregulation of FOXM1 (78%) and TYMS (48%) expression at the protein levels in CCA tissues. In agreement, RT-qPCRand western blot analyses of four human CCA cell lines at the baseline level and in response to high doses of 5-FUrevealed good correlations between FOXM1 and TYMS expression in the CCA cell lines tested, except for the highly 5-FU-resistant HuCCA cells. Consistently, siRNA-mediated knockdown of FOXM1 reduced the clonogenicity and TYMSexpression in the relatively sensitive KKU-D131 but not in the highly resistant HuCCA cells. Interestingly, silencing ofTYMS sensitized both KKU-D131 and HuCCA to 5-FU treatment, suggesting that resistance to very high levels of 5-FU isdue to the inability of the genotoxic sensor FOXM1 to modulate TYMS expression. Consistently, ChIP analysis revealedthat FOXM1 binds efficiently to the TYMS promoter and modulates TYMS expression at the promoter level upon 5-FUtreatment in KKU-D131 but not in HuCCA cells. In addition, E2F1 expression did not correlate with either FOXM1 orTYMS expression and E2F1 depletion has no effects on the clonogenicity and TYMS expression in the CCA cells. Inconclusion, our data show that FOXM1 regulates TYMS expression to modulate 5-FU resistance in CCA and that severe5-FU resistance can be caused by the uncoupling of the regulation of TYMS by FOXM1. Ourfindings suggest that theFOXM1–TYMS axis can be a novel diagnostic, predictive and prognostic marker as well as a therapeutic target for CC