High BRCA1 gene expression increases the risk of early distant metastasis in ER+ breast cancers

Although the function of the BRCA1 gene has been extensively studied, the relationship between BRCA1 gene expression and tumor aggressiveness remains controversial in sporadic breast cancers. Because the BRCA1 protein is known to regulate estrogen signaling, we selected microarray data of ER+ breast cancers from the GEO public repository to resolve previous conflicting findings. The BRCA1 gene expression level in highly proliferative luminal B tumors was shown to be higher than that in luminal A tumors. Survival analysis using a cure model indicated that patients of early ER+ breast cancers with high BRCA1 expression developed rapid distant metastasis. In addition, the proliferation marker genes MKI67 and PCNA, which are characteristic of aggressive tumors, were also highly expressed in patients with high BRCA1 expression. The associations among high BRCA1 expression, high proliferation marker expression, and high risk of distant metastasis emerged in independent datasets, regardless of tamoxifen treatment. Tamoxifen therapy could improve the metastasis-free fraction of high BRCA1 expression patients. Our findings link BRCA1 expression with proliferation and possibly distant metastasis via the ER signaling pathway. We propose a testable hypothesis based on these consistent results and offer an interpretation for our reported associations.

Association between genetic polymorphisms in cytochrome P450 enzymes and survivals in women with breast cancer receiving adjuvant endocrine therapy: a systematic review and meta-analysis

Tamoxifen is commonly prescribed for preventing recurrence in patients with breast cancer. However, the responses of the patients on tamoxifen treatment are variable. Cytochrome P450 genetic variants have been reported to have a significant impact on the clinical outcomes of tamoxifen treatment but no tangible conclusion can be made up till now. The present review attempts to provide a comprehensive review on the associative relationship between genetic polymorphisms in cytochrome P450 enzymes and survival in breast cancer patients on adjuvant tamoxifen therapy. The literature search was conducted using five databases, resulting in the inclusion of 58 studies in the review. An appraisal of the reporting quality of the included studies was conducted using the assessment tool from the Effective Public Health Practice Project (EPHPP). Meta-analyses were performed on CYP2D6 studies using Review Manager 5.3 software. For other studies, descriptive analyses were performed. The results of meta-analyses demonstrated that shorter overall survival, disease-free survival and relapse-free survival were found in the patients with decreased metabolisers when compared to normal metabolisers. The findings also showed that varying and conflicting results were reported by the included studies. The possible explanations for the variable results are discussed in this review.

Conditional Deletion of Pdcd1 Identifies the Cell-Intrinsic Action of PD-1 on Functional CD8 T Cell Subsets for Antitumor Efficacy

Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity.

A Durable Anatomy with Local Plasticity Enables Normal and Stress Hematopoiesis

Knowledge of the anatomy of each tissue and the relationships between progenitors and daughter cells is necessary to understand physiology and pathology. The anatomy of hematopoiesis in the marrow remains largely unknown. Here we identify strategies to image all steps of blood cell production in the mouse sternum using confocal microscopy. We show that long-distance migration of multipotent progenitors, lineage-committed progenitor recruitment to vessels, and generation of lineage-specific oligoclonal structures that are the main production sites for immature cells are key features of the anatomy of blood production. This structural organization is extremely durable and resilient to insults as it was maintained after hemorrhage, Listeria monocytogenes infection, and aging (80-weeks). Importantly, this anatomy is also enabled with local plasticity as production sites for each blood lineage selectively expand/contract in respond to insults followed by a return to homeostasis. We used immunophenotyping to identify fifty-six surface markers that can be combined to image any populations of interest. For example, we found that ESAM is selectively expressed in 100% of LT-HSC, 90% of ST-HSC, 70% of MPP2 and MPP3, 30% of MPP4, 10% of CMP, and 90% of MkP and megakaryocytes but absent in more mature cells. Transplantation experiments revealed that all functional LT- and ST-HSC, MPP2, MPP3 and CMP were contained -exclusively- in the ESAM positive fraction (p<0.05 when compared with ESAM- cells n= 7 mice per group). ESAM + MPP4 displayed 5-fold more engraftment than ESAM - MPP4 (p<0.05). Combining ESAM with classical HSPC markers allowed imaging of all LT-HSC, ST-HSC, MkP, Pre-MegE, MPP2 and a mixed population of MPP3, CMP and MPP4. We developed similar strategies to map erythropoiesis (Pre-MegE → Pre-CFU-E → CFU-E → early erythroblast → late erythroblast → reticulocyte → erythrocyte) and lymphopoiesis (CLP→ PreProB → ProB →PreB→ Immature B). All strategies allowed clonal fate-mapping using Ubc-cre ERT2:confetti mice. In this model tamoxifen treatment leads to irreversible expression of one out of four fluorescent proteins. We found that multipotent and oligopotent progenitors are found as single cells, evenly distributed through the marrow (e.g. mean LT-HSC distance to closest ST-HSC, MPP2, MPP3, MkP, Pre-MegE >100 μm, no different from random simulations, n=41 LT-HSC from 5 sternum sections of 4 wild-type mice) and are clonally unrelated between them. Multipotent and oligopotent progenitors reside near sinusoids (mean distance =9.7 μm) but this association is not different from that observed for random cells. In contrast, as progenitors become lineage-restricted, they localize to arterioles (for lymphoid progenitors) or sinusoids (all other progenitors) where they enter oligoclonal structures that are the main production sites for immature cells in each lineage. Each production site has distinct architectures: lymphoid sites are characterized by tight clusters of PreProB cells surrounding CLP; erythroid sites are characterized by strings of 4-21 CFU-E decorating the surface of sinusoids with early erythroblasts differentiating orthogonally to the vessel surface; in megakaryocyte sites one or two megakaryocyte progenitors produce megakaryocytes that decorate blood vessels over large (>200μm 3) marrow regions. We previously showed that production sites for neutrophils contain 1 or 2 granulocyte progenitors tightly clustered with preneutrophils and that sites for monocytes/dendritic cells contain loose clusters of dendritic cells surrounding monocyte dendritic cell progenitors (Zhang Nature 2021). This spatial architecture is durable and resilient and is maintained after acute challenge via phlebotomy, L. monocytogenes infection, or physiological aging (80-week-old mice). However, we also observed plasticity of production sites. Two days after phlebotomy we found increases in erythroid site numbers (368 vs 945 per mm 3, p<0.05). These expansions were reversed by day 8 after phlebotomy. Similarly, infection led to increases in the size of neutrophil and dendritic cell production sites (~2-fold by day 6 post-infection) but these changes are reverted by day 20 post-infection. In summary, we have developed strategies that allow imaging of differentiation in situ and defined a complex - but durable and plastic- anatomy for the hematopoietic tissue. Disclosures No relevant conflicts of interest to declare.

Mps1 contributes to tamoxifen resistance in Breast Cancer through phosphorylation of ERα

Overexpression of mitotic kinase monopolar spindle 1 (Mps1) has been identified in many tumor types and targeting Mps1 for tumor therapy has been shown great promise in multiple preclinical cancer models. However, the role of Mps1 in tamoxifen resistance in breast cancer has never been reported. Here in this study, we report that Mps1 determined the sensitivity of breast cancer cells to tamoxifen treatment. Mps1 overexpression rendered breast cancer cells more resistant to tamoxifen, while Mps1 inhibitor or siMps1 oligos could overcome tamoxifen resistance. Mechanistically, Mps1 interacted with ERα and stimulated its transcriptional activity in kinase activity-dependent manner. Mps1 was responsible for ERα phosphorylation at S559 and T224 sites. Importantly, Mps1 failed to enhance the transcriptional activity of ERα in the presence of ERα S559A or T224A mutant. Collectively, our findings suggest that Mps1 contributes to tamoxifen resistance in breast cancer and is a potential therapeutic to overcome tamoxifen resistance in breast cancer.

IMMU-22. THE IMPACT OF MICROGLIA MODULATION ON GBM PROGRESSION IN SYNGENEIC MOUSE MODELS

BACKGROUND The tumor immune microenvironment (TME) of Glioblastoma consists of almost myeloid-derived macrophages and microglia called TAMs. We have shown that the disruption of CD47-Sirpα-axis induces an antitumor activity of TAMs against GBM in immune-deficient mice, through increases of phagocytosis of tumor cells by TAMs. We have aimed to study the role of microglia and its activation/depletion on GBM progression, in the syngeneic GBM model in immune-competent mice. We have studied the interplay of innate and adaptive immune response after activation and depletion of microglia and the effect on tumor progression and outcome of the mice. MATERIAL AND METHODS We used different colonies of genetically modified immunocompetent mouse strains to genetically activate/deplete microglia in the tumor context. We generated Sall1 CreERT2/fl mice and Cre-negative littermates. The application of Tamoxifen in this constellation leads to the excision of the transcription factor Sall1 and subsequent enhanced microglia activity. Conversely, we generated Sall1 CreERT2 x Csf1r fl/fl animals and the respective heterozygous and Cre-negative littermates in which Tamoxifen treatment leads to inactivation of microglia through the deletion of Csf1r. Glioblastoma tumors were induced by intracerebral injection of GL261, CT2A, or retrovirus-induced PDGF-Akt in pups and Tamoxifen treatment was started once the tumors were detected. RESULTS We observed a survival advantage in tumor-bearing mice after activation of microglia in Sall1 CreERT/fl animals compared to Cre-negative littermates. Genetic depletion of microglia in this model resulted in a shorter lifespan in microglia-depleted animals compared to Cre-negative littermates. Furthermore, the iTME in these tumors is subjected to scRNAseq analysis to identify mechanistic insights. CONCLUSION Microglia are important players in tumor development and progression of glioblastoma in mouse models. These cells may be targeted in future immunotherapeutic approaches for patients.

Molecular markers associated with the outcome of tamoxifen treatment in estrogen receptor-positive breast cancer patients: scoping review and in silico analysis

Tamoxifen (TMX) is used as adjuvant therapy for estrogen receptor-positive (ER+) breast cancer cases due to its affinity and inhibitory effects. However, about 30% of cases show drug resistance, resulting in recurrence and metastasis, the leading causes of death. A literature review can help to elucidate the main cellular processes involved in TMX resistance. A scoping review was performed to find clinical studies investigating the association of expression of molecular markers profiles with long-term outcomes in ER+ patients treated with TMX. In silico analysis was performed to assess the interrelationship among the selected markers, evaluating the joint involvement with the biological processes. Forty-five studies were selected according to the inclusion and exclusion criteria. After clustering and gene ontology analysis, 23 molecular markers were significantly associated, forming three clusters of strong correlation with cell cycle regulation, signal transduction of proliferative stimuli, and hormone response involved in morphogenesis and differentiation of mammary gland. Also, it was found that overexpression of markers in selected clusters is a significant indicator of poor overall survival. The proposed review offered a better understanding of independent data from the literature, revealing an integrative network of markers involved in cellular processes that could modulate the response of TMX. Analysis of these mechanisms and their molecular components could improve the effectiveness of TMX.

Kinetics of alpha-synuclein depletion in three brain regions following conditional pan-neuronal inactivation of the encoding gene (Snca) by tamoxifen-induced Cre-recombination in adult mice

Conditional pan-neuronal inactivation of the Snca gene in 2-month old male and female mice causes dramatic decrease in the level of the encoded protein, alpha-synuclein, in three studied brain regions, namely cerebral cortex, midbrain and striatum, 12 weeks after the last injection of tamoxifen. Kinetics of alpha-synuclein depletion is different in these brain regions with a longer lag period in the cerebral cortex where this protein is normally most abundant. Our results suggest that efficient post-developmental pan-neuronal knockout of alpha-synuclein in adult, i.e. 5- to 6-month old, animals, could be achieved by tamoxifen treatment of 2-month old mice carrying loxP-flanked Snca gene and expressing inducible Cre-ERT2 recombinase under control of the promoter of neuron-specific enolase (NSE) gene.

Tamoxifen administration induces histopathologic changes within the lungs of Cre-recombinase-negative mice: A case report

Tamoxifen is commonly used as a cancer treatment in humans and for inducing genetic alterations using Cre-lox mouse models in the research setting. However, the extent of tamoxifen off-target effects in animal research is underappreciated. Here, we report significant changes in cellular infiltration in Cre-recombinase-negative mice treated with tamoxifen intraperitoneally. These changes were noted in the lungs, which were characterized by the presence of alveolitis, vasculitis, and pleuritis. Despite significant immunological changes in response to tamoxifen treatment, clinical symptoms were not observed. This study provides a cautionary note that tamoxifen treatment alone leads to histologic alterations that may obscure research interpretations and further highlights the need for the development of alternative mouse models for inducible Cre-mediated deletion.