Oncological outcomes post transoral robotic surgery (TORS) for HPV-associated oropharyngeal squamous cell carcinoma, a single-centre retrospective Australian study.

We present a cohort review of TORS resection for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) and its associated oncological outcomes spanning a 10-year period. A retrospective case series review was performed of patients undergoing primary surgical treatment for HPV-associated OPSCC through the St. Vincent's Head and Neck Cancer service from 2011 to 2022. The primary outcomes were to investigate complete resection of the primary tumour, rates of recurrence, and survival analysis. Secondary outcomes included complications, rates of adjuvant therapy, sites of recurrence and rates of percutaneous endoscopic gastrostomy (PEG). 184 patients underwent TORS-based therapy with neck dissection, and guideline-directed adjuvant therapy for HPV-associated OPSCC. Our median follow-up was 46 months. The positive margin rate on final histopathology analysis was 10.9%. Adjuvant therapy was indicated in 85 patients (46%). The local recurrence rate was 10.9% with the majority (80%) of patients recurring in the first 3 years since treatment. The disease-specific survival at 3 years was 98.6% and at 5 years was 94.4%. The 3-year and 5-year OS for the cohort was 96.7% and 92.5%, respectively. The presence of extranodal extension and positive margins were associated with increased risk of recurrence, whereas adjuvant therapy was found to be a protective factor for both overall recurrence and survival. Major complications occurred in 12 patients (6.5%), resulting in one death. This study has demonstrated that primary surgical therapy for HPV-associated OPSCC is a safe and effective treatment modality with low local recurrence and complication rates, and overall survival benefits.

SILAC kinase screen identifies potential MASTL substrates.

Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.

Activin-A, Growth Differentiation Factor-11 and Transforming Growth Factor-ß as predictive biomarkers for platinum chemotherapy in advanced non-small cell lung cancer.

BACKGROUND: Despite advances in immunotherapy and targeted therapy, platinum-based chemotherapy remains crucial for many patients with advanced non-small cell lung cancer (NSCLC). Resistance to platinum chemotherapy is common, and predictive biomarkers are needed to tailor treatment to patients likely to respond. In vitro evidence implicates the transforming growth factor-ß (TGF-ß) superfamily ligands activin-A and growth differentiation factor 11 (GDF-11) in innate platinum resistance. We performed a validation study to assess their utility as predictive biomarkers of platinum chemotherapy response in advanced NSCLC. PATIENTS AND METHODS: Our study included 123 adult patients with advanced NSCLC without a driver mutation treated with platinum chemotherapy. 98 patients were from a retrospective cohort and 25 from a prospective cohort. We performed immunohistochemistry staining for Activin-A, GDF-11 and TGF-ß on tumour samples for each patient and analysed IHC expression with objective radiological response and overall survival. RESULTS: The overall median survival was 14.8 months. We performed statistical analysis around a cytoplasmic score of 8/18 for Activin-A and GDF-11 based on previously published work, and 110/30 for TGF-ß based on a calculated cutpoint for significance. No survival difference was detected between these groups for Activin-A (p=0.35), GDF-11 (p=0.57) or TGF-ß (p=0.34). There was no association between rates of progressive disease and high Activin-A expression (p=0.43), high GDF-11 expression (p=1.0) or high TGF-ß expression p=0.89). CONCLUSION: Within the confines of our study, Activin-A, GDF-11 and TGF-ß expression was not a predictor of objective radiological response to chemotherapy or overall survival.

A non-genetic, cell cycle-dependent mechanism of platinum resistance in lung adenocarcinoma.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

Altered presentation of oropharyngeal cancer, a 6-year review.

BACKGROUND: Human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) continues to increase in incidence. Patients are younger, non-smokers and most commonly present with a neck mass often with no other symptoms. This altered presentation compared with non-HPV OPSCC may not be recognized by medical practitioners, leading to delayed diagnosis. METHODS: Patients with histopathological confirmation of OPSCC and known HPV and/or P16 status who presented to our institution between 2012-2017 inclusive were included in the study. Demographic data, tumour characteristics and presenting symptoms were retrospectivxely obtained from both electronic- and paper-based records. Descriptive statistics were used to report demographic data and the two sample t-test and Fisher's exact test were used to compare groups based on HPV status. Time to diagnosis was also reported. RESULTS: A total of 184 patients were included in the study. The majority of patients were male (85.4%) and HPV + (85.3%). The tonsillar complex (53.8%) and tongue base (42.4%) were the most common primary sites. HPV+ patients were less likely to smoke (17.8%) and they commonly presented with a neck mass (39.5% alone or with other symptoms 61.2%). Time to diagnosis in the HPV+ group was longer (15 weeks). CONCLUSION: Our review has highlighted the altered presentation of OPSCC due to the increased incidence of HPV infection. We showed a delayed time to diagnosis in HPV+ OPSCC compared with non-HPV disease. This confirms the importance of focusing our efforts on educating medical practitioners and creating further awareness to facilitate early detection and treatment.

Analysis of pulsed cisplatin signalling dynamics identifies effectors of resistance in lung adenocarcinoma.

The identification of clinically viable strategies for overcoming resistance to platinum chemotherapy in lung adenocarcinoma has previously been hampered by inappropriately tailored in vitro assays of drug response. Therefore, using a pulse model that closely mimics the in vivo pharmacokinetics of platinum therapy, we profiled cisplatin-induced signalling, DNA-damage and apoptotic responses across a panel of human lung adenocarcinoma cell lines. By coupling this data to real-time, single-cell imaging of cell cycle and apoptosis we provide a fine-grained stratification of response, where a P70S6K-mediated signalling axis promotes resistance on a TP53 wildtype or null background, but not a mutant TP53 background. This finding highlights the value of in vitro models that match the physiological pharmacokinetics of drug exposure. Furthermore, it also demonstrates the importance of a mechanistic understanding of the interplay between somatic mutations and the signalling networks that govern drug response for the implementation of any consistently effective, patient-specific therapy.

Lung adenocarcinoma is the most common type of lung cancer, and it emerges because of a variety of harmful genetic changes, or mutations. Two lung cancer patients ­ or indeed, two different sets of cancerous cells within a patient ­ may therefore carry different damaging mutations. A group of drugs called platinum-based chemotherapies are currently the most effective way to treat lung adenocarcinoma. Yet, only 30% of patients actually respond to the therapy. Many studies conducted in laboratory settings have tried to understand why most cases are resistant to treatment, with limited success. Here, Hastings, Gonzalez-Rajal et al. propose that previous research has been inconclusive because studies done in the laboratory do not reflect how the treatment is actually administered. In patients, platinum-based drugs are cleared from the body within a few hours, but during experiments, the treatment is continually administered to cells growing in a dish. Hastings, Gonzalez-Rajal et al. therefore developed a laboratory method that mimics the way cells are exposed to platinum-based chemotherapy in the body. These experiments showed that the lung adenocarcinoma cells which resisted treatment also carried high levels of a protein known as P70S6K. Pairing platinum-based chemotherapy with a drug that blocks the activity of P70S6K killed these resistant cells. This combination also treated human lung adenocarcinoma tumours growing under the skin of mice. However, it was ineffective on cancerous cells that carry a mutation in a protein called p53, which is often defective in cancers. Overall, this work demonstrates the need to refine how drugs are tested in the laboratory to better reflect real-life conditions. It also underlines the importance of personalizing drug combinations to the genetic background of each tumour, a concept that will be vital to consider in future clinical trials.

Deep multi-region whole-genome sequencing reveals heterogeneity and gene-by-environment interactions in treatment-naive, metastatic lung cancer.

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy.

Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

MASTL overexpression promotes chromosome instability and metastasis in breast cancer.

MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial-mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.

The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia.

Primary cilia are crucial for signal transduction in a variety of pathways, including hedgehog and Wnt. Disruption of primary cilia formation (ciliogenesis) is linked to numerous developmental disorders (known as ciliopathies) and diseases, including cancer. The ubiquitin-proteasome system (UPS) component UBR5 was previously identified as a putative positive regulator of ciliogenesis in a functional genomics screen. UBR5 is an E3 ubiquitin ligase that is frequently deregulated in tumors, but its biological role in cancer is largely uncharacterized, partly due to a lack of understanding of interacting proteins and pathways. We validated the effect of UBR5 depletion on primary cilia formation using a robust model of ciliogenesis, and identified CSPP1, a centrosomal and ciliary protein required for cilia formation, as a UBR5-interacting protein. We show that UBR5 ubiquitylates CSPP1, and that UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery, suggesting that UBR5-mediated ubiquitylation of CSPP1 or associated centriolar satellite constituents is one underlying requirement for cilia expression. Hence, we have established a key role for UBR5 in ciliogenesis that may have important implications in understanding cancer pathophysiology.

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53.

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis.

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.

PP1 initiates the dephosphorylation of MASTL, triggering mitotic exit and bistability in human cells.

Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit.

Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events.

Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G 2 phase onwards. Addition of low doses of RO3306 in G 2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G 2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G 2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.

BCL-2 hypermethylation is a potential biomarker of sensitivity to antimitotic chemotherapy in endocrine-resistant breast cancer.

Overexpression of the antiapoptotic factor BCL-2 is a frequent feature of malignant disease and is commonly associated with poor prognosis and resistance to conventional chemotherapy. In breast cancer, however, high BCL-2 expression is associated with favorable prognosis, estrogen receptor (ER) positivity, and low tumor grade, whereas low expression is included in several molecular signatures associated with resistance to endocrine therapy. In the present study, we correlate BCL-2 expression and DNA methylation profiles in human breast cancer and in multiple cell models of acquired endocrine resistance to determine whether BCL-2 hypermethylation could provide a useful biomarker of response to cytotoxic therapy. In human disease, diminished expression of BCL-2 was associated with hypermethylation of the second exon, in a region that overlapped a CpG island and an ER-binding site. Hypermethylation of this region, which occurred in 10% of primary tumors, provided a stronger predictor of patient survival (P = 0.019) when compared with gene expression (n = 522). In multiple cell models of acquired endocrine resistance, BCL-2 expression was significantly reduced in parallel with increased DNA methylation of the exon 2 region. The reduction of BCL-2 expression in endocrine-resistant cells lowered their apoptotic threshold to antimitotic agents: nocodazole, paclitaxel, and the PLK1 inhibitor BI2536. This phenomenon could be reversed with ectopic expression of BCL-2, and rescued with the BCL-2 inhibitor ABT-737. Collectively, these data imply that BCL-2 hypermethylation provides a robust biomarker of response to current and next-generation cytotoxic agents in endocrine-resistant breast cancer, which may prove beneficial in directing therapeutic strategy for patients with nonresectable, metastatic disease.

Global characterization of signalling networks associated with tamoxifen resistance in breast cancer.

Acquired resistance to the anti-estrogen tamoxifen remains a significant challenge in breast cancer management. In this study, we used an integrative approach to characterize global protein expression and tyrosine phosphorylation events in tamoxifen-resistant MCF7 breast cancer cells (TamR) compared with parental controls. Quantitative mass spectrometry and computational approaches were combined to identify perturbed signalling networks, and candidate regulatory proteins were functionally interrogated by siRNA-mediated knockdown. Network analysis revealed that cellular metabolism was perturbed in TamR cells, together with pathways enriched for proteins associated with growth factor, cell-cell and cell matrix-initiated signalling. Consistent with known roles for Ras/MAPK and PI3-kinase signalling in tamoxifen resistance, tyrosine-phosphorylated MAPK1, SHC1 and PIK3R2 were elevated in TamR cells. Phosphorylation of the tyrosine kinase Yes and expression of the actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) were increased two- and eightfold in TamR cells respectively, and these proteins were selected for further analysis. Knockdown of either protein in TamR cells had no effect on anti-estrogen sensitivity, but significantly decreased cell motility. MARCKS expression was significantly higher in breast cancer cell lines than normal mammary epithelial cells and in ER-negative versus ER-positive breast cancer cell lines. In primary breast cancers, cytoplasmic MARCKS staining was significantly higher in basal-like and HER2 cancers than in luminal cancers, and was independently predictive of poor survival in multivariate analyses of the whole cohort (P < 0.0001) and in ER-positive patients (P = 0.0005). These findings provide network-level insights into the molecular alterations associated with the tamoxifen-resistant phenotype, and identify MARCKS as a potential biomarker of therapeutic responsiveness that may assist in stratification of patients for optimal therapy.

Tamoxifen-induced epigenetic silencing of oestrogen-regulated genes in anti-hormone resistant breast cancer.

In the present study, we have taken the novel approach of using an in vitro model representative of tamoxifen-withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant-cell phenotype, independent of any agonistic impact of tamoxifen, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. These data provide evidence to support a novel concept that long-term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen-responsive genes whose function is associated with negative proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.