Regulation of PERK expression by FOXO3: a vulnerability of drug-resistant cancer cells

The major impediment to effective cancer therapy has been the development of drug resistance. The tumour suppressive transcription factor FOXO3 promotes cell cycle arrest, senescence and cell death, and mediates the cytotoxic and cytostatic functions of cancer therapeutics. In consequence, FOXO3 is often downregulated as an adaptive response in cancer and particularly in chemotherapeutic drug-resistant cells. Consistently, we find that FOXO3 expression is attenuated in the drug-resistant MCF-7-EpiR and MCF-7-TaxR compared to the parental MCF-7 breast cancer cells. Using ChIP, short-interfering RNA (siRNA) knockdown, and overexpression assays as well as Foxo1/3/4−/− MEFs, we establish the endoplasmic reticulum (ER)-stress defence modulator PERK (eIF2AK3) as a direct downstream transcriptional target of FOXO3. In agreement, there is also a positive correlation between FOXO3 and PERK expression at the protein and RNA levels in breast cancer patient samples. We uncover that PERK expression is downregulated but its activity constitutively elevated in the drug-resistant cells. With this in mind, we exploit this adaptive response of low FOXO3 and PERK expression, and high PERK activity in drug-resistant breast cancer cells and show that these drug-resistant cells are specifically sensitive to PERK inhibition. In support of this finding, we show that ectopic overexpression of FOXO3 can reduce the sensitivity of the resistant cells to the PERK inhibitor GSK2606414, while the Foxo1/3/4−/− MEFs expressing lower levels of PERK are more sensitive to PERK inhibition compared to wild-type MEFs. PERK inhibitor-titration and -time course experiments showed that the drug-resistant cells, which express lower expression and higher activity levels of PERK, are more sensitive to the increasing concentrations of PERK inhibitor compared to parental MCF-7 cells. Our present work thus reveals a chemotherapeutic drug-resistant cancer cell vulnerability in PERK and suggests PERK as a potential target for cancer therapy, specifically in the context of drug-resistant cancers

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

Lapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylation [version 3]

Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666-1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1)are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10Band resistant 5-8FNPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 −/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells

NEDDylation negatively regulates ERRβ expression to promote breast cancer tumorigenesis and progression

Estrogen related receptor beta (ERRβ) is down-regulated in breast cancer cells and its over expression in breast cancer patients is positively correlated with an improved prognosis and prolonged relapse-free survival. Here, we unravelled a molecular mechanism for ERRβ down-regulation in breast cancer. We found that ERRβ is a key substrate of the SCF complex and that NEDDylation can activate the Cullin sub units of the SCF complex to target ERRβfor degradation in breast cancer. Consistently, using in vitro and in vivo models, we demonstrated that MLN4924, a specific small molecule inhibitor of NEDDylation, can restore ERRβ expressionand culminate in a reduction in cell proliferation and migration of breast cancer cells.We also showed that increased ERRβ expression promotes the up-regulation of its 34target genes, including the tumour suppressors p21Cip1/Waf1and E-cadherin,involved in cell proliferation and migration arrest at the gene promoter level. Interestingly, this tumour suppressive role of ERRβ does not depend on the expression of ERain breast cancer. Moreover, our data revealed that the ERRβ recruits the transcription co-activator p300 to its targeted gene promoters to up regulate their expression. Collectively,our work revealed that restoration of ERRβ expression using the NEDDylation inhibitor MLN4924 can be a novel and effective strategy for breast cancer treatmen

EP300 and SIRT1/6 co-regulate lapatinib sensitivity via modulating FOXO3-acetylation and activity in breast cancer

Forkhead Box O3 (FOXO3) is a tumour suppressor whose activity is fine-tuned by post-translational modifications (PTMs). In this study, using the BT474 breast cancer cells and a recently established lapatinib resistant (BT474-LapR) cell line, we observed that higher FOXO3 and acetylated (Ac)-FOXO3 levels correlate with lapatinib sensitivity. Subsequent ectopic expression of EP300 led to an increase in acetylated-FOXO3 in sensitive, but not in resistant cells. Drug sensitivity assays revealed that sensitive BT474 cells show increased lapatinib cytotoxicity upon over-expression of wild-type but not acetylation-deficient EP300. Moreover, FOXO3 recruitment to target gene promoters is associated with target gene expression and drug response in sensitive cells, and the inability of FOXO3 to bind its target genes correlates with lapatinib-resistance in BT474-LapR cells. In addition, using SIRT1/6 specific siRNAs and chemical inhibitor, we also found that sirtuin 1 and -6 (SIRT1 and -6) play a part in fine-tuning FOXO3 acetylation and lapatinib sensitivity. Consistent with this, immunohistochemistry results from different breast cancer subtypes showed that high SIRT6/1 levels are associated with constitutive high FOXO3 expression which is related to FOXO3 deregulation/inactivation and poor prognosis in breast cancer patient samples. Collectively, our results suggest the involvement of FOXO3 acetylation in regulating lapatinib sensitivity of HER2-positive breast cancers

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