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.

The endoplasmic reticulum (ER) is a cellular organelle with central roles in maintaining proteostasis due to its involvement in protein synthesis, folding, quality control, distribution and degradation. The accumulation of misfolded proteins in the ER lumen causes 'ER stress' and threatens overall cellular proteostasis. To restore ER homeostasis, cells evoke an evolutionarily conserved adaptive signalling and gene expression network collectively called the 'unfolded protein response (UPR)', a complex biological process which aims to restore proteostasis. When ER stress is overwhelming and beyond rectification, the normally pro-survival UPR can shift to induce cell termination. Emerging evidence from mammalian, fly and nematode worm systems reveals that the FOXO Forkhead proteins integrate upstream ER stress and UPR signals with the transcriptional machinery to decrease translation, promote cell survival/termination and increase the levels of ER-resident chaperones and of ER-associated degradation (ERAD) components to restore ER homeostasis. The high rates of protein synthesis/translation associated with cancer cell proliferation and metabolism, as well as mutations resulting in aberrant proteins, also induce ER stress and the UPR. While the pro-survival side of the UPR underlies its ability to sustain and promote cancers, its apoptotic functions can be exploited for cancer therapies by offering the chance to 'flick the proteostatic switch'. To this end, further studies are required to fully reevaluate the roles and regulation of these UPR signalling molecules, including FOXO proteins and their targets, in cancer initiation and progression as well as the effects on inhibiting their functions in cancer cells. This information will help to establish these UPR signalling molecules as possible therapeutic targets and putative biomarkers in cancers.

Forkhead Box O3 (FOXO3) is a tumor 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.