Hormone dependent breast cancer (HDBC) is the most commonly diagnosed tumor type in women. Adjuvant endocrine therapies (ET) have been the cornerstone in the clinical management of HDBC patients for over forty years. A vast proportion of HDBC patients incur long periods of clinical dormancy following ET, with tumour awakening appearing at a steady pace for up to 25 years (Pan et al., 2017). Extensive genomic studies have demonstrated that 15-30% of clinical relapses develop recurrent genomic changes which contribute to drug resistance (i.e. ESR1 activating mutations) (Bertucci et al., 2019; Magnani et al., 2017; Razavi et al., 2018). However, even in these cases, there is no conclusive evidence around the pre-existence vs. de novo nature of these events. We previously showed that ETs can trigger and select for dormancy in subpopulations of breast cancer (Hong et al., 2019). In this work we took two novel approaches to investigate the dormancy and awakening roadmap of HDBC cells at unprecedented detail. Firstly, we leveraged a rare cohort of n=5 patients which were treated with primary adjuvant ETs in the absence of surgery (TRACING-HT) to dissect the contribution of genomic aberrations to tumor awakening. Next, we developed a first of its kind evolutionary study in vitro to systematically annotate cancer cells adaptive strategies at single cell level in unperturbed systems during a period of several months (TRADITIOM). Collectively our data suggest that ETs steer HDBC cells into an inherently unstable dormant state. Over time, routes to awakening emerge sporadically and spontaneously in single lineages. Each dormant cell retains an intrinsic awakening probability which we propose is a function of epigenetic decay. Awakening occurs without an external trigger and involves multiple apparent endpoint phenotypes that cannot be fully explained by conventional Darwinian genetic selection processes. Finally, our data show that common genetic hits associated with resistance happen downstream of awakening. Overall, our data have uncovered previously unsuspected roles for stochastic nongenetic events during dormancy with profound clinical implications.

Background/Aims: Helicobacter pylori (H. pylori) is a microaerophilic bacterium related with peptic ulcer and gastric cancer. Its virulence factors include cytotoxin-associated gene A (CagA) and vacuolating cytotoxin gene A (VacA) proteins. Cytokine release inducted by H. pylori colonization has an important role in pathogenesis of H. pylori. The severity of gastric pathologies depends on the H. pylori genotypes found in different geographical regions. We aimed to determine the relationship between different H. pylori genotypes and their effects on the cytokine release levels. Materials and Methods: ureC, cagA, vacAs1/s2, vacAm1/m2, and blood group antigen-binding adhesion protein A2 (babA2) virulence related genes were investigated in 21 H. pylori strains. Genotyping of 21 strains were made due to the presence of cagA, vacAs1/s2, vacAm1/m2, and babA2 genes. The H. pylori strains were cultured together with THP-1 and neutrophil-differentiated Human promyelocytic leukemia cells (HL-60) cells. The levels of cytokines interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor-alpha (TNF-α), and IL-10 in these cells were measured after co-culturing with H. pylori strains. Results: The following five different genotypes were detected: Genotype1: cagA and vacAs1m2; Genotype2: cagA and vacAs1m1; Genotype3: cagA, vacAs1m2, and babA2; Genotype4: vacAs2m2; and Genotype5: cagA and vacAs2m2. All these genotypes significantly induced the levels of IL-1β, IL-6, IL-8, IL 10, and TNF-α in THP-1 cells. Genotype 5 caused higher amounts of IL-1β, IL-6, TNF-α, and IL-10, whereas genotype 1 induced the highest levels of IL-8. In neutrophil-differentiated HL-60 cells, genotype 4 increased IL-6 levels and genotype 3 and 4 elevated IL-8 levels significantly. Conclusion: These results suggested that cytokine response of the host varies depending on the specific immune response of the host against different H. pylori strains.

Background Estrogen receptor (ER) positive breast cancer is often effectively treated with drugs that inhibit ER signaling, i.e., tamoxifen (TAM) and aromatase inhibitors (AIs). However, 30% of ER+ breast cancer patients develop resistance to therapy leading to tumour recurrence. Changes in the methylation profile have been implicated as one of the mechanisms through which therapy resistance develops. Therefore, we aimed to identify methylation loci associated with endocrine therapy resistance. Methods We used genome-wide DNA methylation profiles of primary ER+/HER2- tumours from The Cancer Genome Atlas in combination with curated data on survival and treatment to predict development of endocrine resistance. Association of individual DNA methylation markers with survival was assessed using Cox proportional hazards models in a cohort of ER+/HER2- tumours ( N = 552) and two sub-cohorts corresponding to the endocrine treatment (AI or TAM) that patients received ( N = 210 and N = 172, respectively). We also identified multivariable methylation signatures associated with survival using Cox proportional hazards models with elastic net regularization. Individual markers and multivariable signatures were compared with DNA methylation profiles generated in a time course experiment using the T47D ER+ breast cancer cell line treated with tamoxifen or deprived from estrogen. Results We identified 134, 5 and 1 CpGs for which DNA methylation is significantly associated with survival in the ER+/HER2-, TAM and AI cohorts respectively. Multi-locus signatures consisted of 203, 36 and 178 CpGs and showed a large overlap with the corresponding single-locus signatures. The methylation signatures were associated with survival independently of tumour stage, age, AI treatment, and luminal status. The single-locus signature for the TAM cohort was conserved among the loci that were differentially methylated in endocrine-resistant T47D cells. Similarly, multi-locus signatures for the ER+/HER2- and AI cohorts were conserved in endocrine-resistant T47D cells. Also at the gene set level, several sets related to endocrine therapy and resistance were enriched in both survival and T47D signatures. Conclusions We identified individual and multivariable DNA methylation markers associated with therapy resistance independently of luminal status. Our results suggest that these markers identified from primary tumours prior to endocrine treatment are associated with development of endocrine resistance.

Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor α account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, depending on the degree of severity of the disease at diagnosis, 10 to 40% of these tumors eventually relapse due to resistance development. Even though recent novel approaches as the combination with CDK4/6 inhibitors increased the overall survival of relapsing patients, this remains relatively short and there is a urgent need to find alternative targetable pathways. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle, and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model, and elevated expression of ATF3 was verified in patient datasets, adding clinical relevance to our findings. This study proposes ATF3 as a novel mediator of endocrine resistance development in breast cancer and elucidates its role in the regulation of downstream pathways activities.