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Figure S1. Cellular senescence, expression profiling and the SASP development in human stromal cells upon genotoxic treatment. Figure S2. SIRT1 decline is not related with single strand breaks, proteasome degradation or autophagy deficiency upon cellular senescence. Figure S3. Senescent stromal sEVs enhance the malignancy of prostate cancer cells. Figure S4. Transcriptomic profiling of prostate cancer cells upon exposure to senescent stromal sEVs and establishment of protein-protein interaction network involving ABCB4. Figure S5. Senescent stromal sEV-conferred malignancy is diminished upon elimination of ABCB4 from cancer cells. Figure S6. Pharmacological targeting SIRT1 minimizes senescent stromal sEV production and prevents ABCB4 upregulation in prostate cancer cells.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype, which is implicated in age-related pathologies including cancer. Here, we report that senescent cells actively synthesize and release small extracellular vesicles (sEV) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP-binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathologic activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by cotargeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells.Significance:Senescent stromal cells produce a large number of sEVs to promote cancer resistance in therapeutic settings, a process driven by SIRT1 decline in stromal cells and ABCB4 augmentation in cancer cells.See related commentary by Wiley, p. 3193

Fluorouracil (5-FU) is the first-line chemotherapeutic drug for cholangiocarcinoma (CCA), but its efficacy has been compromised by the development of resistance. Development of 5-FU resistance is associated with elevated expression of its cellular target, thymidylate synthase (TYMS). E2F1 transcription factor has previously been shown to modulate the expression of FOXM1 and TYMS. Immunohistochemical (IHC) analysis revealed a strong correlated upregulation of FOXM1 (78%) and TYMS (48%) expression at the protein levels in CCA tissues. In agreement, RT-qPCR and western blot analyses of four human CCA cell lines at the baseline level and in response to high doses of 5-FU revealed 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 TYMS expression in the relatively sensitive KKU-D131 but not in the highly resistant HuCCA cells. Interestingly, silencing of TYMS sensitized both KKU-D131 and HuCCA to 5-FU treatment, suggesting that resistance to very high levels of 5-FU is due to the inability of the genotoxic sensor FOXM1 to modulate TYMS expression. Consistently, ChIP analysis revealed that FOXM1 binds efficiently to the TYMS promoter and modulates TYMS expression at the promoter level upon 5-FU treatment in KKU-D131 but not in HuCCA cells. In addition, E2F1 expression did not correlate with either FOXM1 or TYMS expression and E2F1 depletion has no effects on the clonogenicity and TYMS expression in the CCA cells. In conclusion, our data show that FOXM1 regulates TYMS expression to modulate 5-FU resistance in CCA and that severe 5-FU resistance can be caused by the uncoupling of the regulation of TYMS by FOXM1. Our findings suggest that the FOXM1–TYMS axis can be a novel diagnostic, predictive and prognostic marker as well as a therapeutic target for CCA.

Estrogen-related receptor beta (ERRβ) is downregulated in breast cancer cells and its overexpression in breast cancer patients is positively correlated with an improved prognosis and prolonged relapse-free survival. Here, we unravelled a molecular mechanism for ERRβ downregulation in breast cancer. We found that ERRβ is a key substrate of the SCF complex and that NEDDylation can activate the Cullin subunits of the SCF complex to target ERRβ for 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 expression and culminate in a reduction in cell proliferation and migration of breast cancer cells. We also showed that increased ERRβ expression promotes the upregulation of its target genes, including the tumour suppressors p21Cip1/Waf1 and 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 ERα in breast cancer. Moreover, our data revealed that the ERRβ recruits the transcription co-activator p300 to its targeted gene promoters to upregulate 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 treatment.

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., Highlights • Compensatory mechanisms compromise PERK (eIF2AK3) inhibitor response. • GCN2 (eIF2AK4) cooperates with PERK to modulate cancer drug response. • GCN2 and PERK regulate FOXO3 via JNK and AKT in response to cancer drug. • GCN2 regulates PERK and vice versa in a reciprocal negative feedback loop. • Inhibiting GCN2 is a novel strategy for targeting cancer and drug resistance.

Forkhead box O (FOXO) transcription factors regulate diverse biological processes, affecting development, metabolism, stem cell maintenance and longevity. They have also been increasingly recognised as tumour suppressors through their ability to regulate genes essential for cell proliferation, cell death, senescence, angiogenesis, cell migration and metastasis. Mechanistically, FOXO proteins serve as key connection points to allow diverse proliferative, nutrient and stress signals to converge and integrate with distinct gene networks to control cell fate, metabolism and cancer development. In consequence, deregulation of FOXO expression and function can promote genetic disorders, metabolic diseases, deregulated ageing and cancer. Metastasis is the process by which cancer cells spread from the primary tumour often via the bloodstream or the lymphatic system and is the major cause of cancer death. The regulation and deregulation of FOXO transcription factors occur predominantly at the post-transcriptional and post-translational levels mediated by regulatory non-coding RNAs, their interactions with other protein partners and co-factors and a combination of post-translational modifications (PTMs), including phosphorylation, acetylation, methylation and ubiquitination. This review discusses the role and regulation of FOXO proteins in tumour initiation and progression, with a particular emphasis on cancer metastasis. An understanding of how signalling networks integrate with the FOXO transcription factors to modulate their developmental, metabolic and tumour-suppressive functions in normal tissues and in cancer will offer a new perspective on tumorigenesis and metastasis, and open up therapeutic opportunities for malignant diseases.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype, which is implicated in age-related pathologies including cancer. Here, we report that senescent cells actively synthesize and release small extracellular vesicles (sEV) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP-binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathologic activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by cotargeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. Significance: Senescent stromal cells produce a large number of sEVs to promote cancer resistance in therapeutic settings, a process driven by SIRT1 decline in stromal cells and ABCB4 augmentation in cancer cells. See related commentary by Wiley, p. 3193

Obesity has for decades been recognised as one of the major health concerns. Recently accumulated evidence has established that obesity or being overweight is strongly linked to an increased risk of cancer. However, it is still not completely clear how adipose tissue (fat), along with other stromal connective tissues and cells, contribute to tumour initiation and progression. In the tumour microenvironment, the adipose tissue cells, in particular the adipocytes, secrete a number of adipokines, including growth factors, hormones, collagens, fatty acids, and other metabolites as well as extracellular vesicles to shape and condition the tumour and its microenvironment. In fact, the adipocytes, through releasing these factors and materials, can directly and indirectly facilitate cancer cell proliferation, apoptosis, metabolism, angiogenesis, metastasis and even chemotherapy resistance. In this chapter, the multidimensional role played by adipocytes, a major and functional component of the adipose tissue, in promoting cancer development and progression within the tumour microenvironment will be discussed.

Obesity has for decades been recognised as one of the major health concerns. Recently accumulated evidence has established that obesity or being overweight is strongly linked to an increased risk of cancer. However, it is still not completely clear how adipose tissue (fat), along with other stromal connective tissues and cells, contribute to tumour initiation and progression. In the tumour microenvironment, the adipose tissue cells, in particular the adipocytes, secrete a number of adipokines, including growth factors, hormones, collagens, fatty acids, and other metabolites as well as extracellular vesicles to shape and condition the tumour and its microenvironment. In fact, the adipocytes, through releasing these factors and materials, can directly and indirectly facilitate cancer cell proliferation, apoptosis, metabolism, angiogenesis, metastasis and even chemotherapy resistance. In this chapter, the multidimensional role played by adipocytes, a major and functional component of the adipose tissue, in promoting cancer development and progression within the tumour microenvironment will be discussed.

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 6-10B and resistant 5-8F NPC 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. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.

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.