The tumor immune microenvironment dynamically evolves to support tumor growth and progression. Immunosuppressive regulatory T cells (Treg) promote tumor growth and metastatic seeding in patients with breast cancer. Deregulation of plasticity between Treg and Th17 cells creates an immune regulatory framework that enables tumor progression. Here, we discovered a functional role for Hedgehog (Hh) signaling in promoting Treg differentiation and immunosuppressive activity, and when Hh activity was inhibited, Tregs adopted a Th17-like phenotype complemented by an enhanced inflammatory profile. Mechanistically, Hh signaling promoted O-GlcNAc modifications of critical Treg and Th17 transcription factors, Foxp3 and STAT3, respectively, that orchestrated this transition. Blocking Hh reprogramed Tregs metabolically, dampened their immunosuppressive activity, and supported their transdifferentiation into inflammatory Th17 cells that enhanced the recruitment of cytotoxic CD8+ T cells into tumors. Our results demonstrate a previously unknown role for Hh signaling in the regulation of Treg differentiation and activity and the switch between Tregs and Th17 cells in the tumor microenvironment.

Macrophages are important cellular components of the innate immune system, serving as the first line of immune defense. They are also among the first immune cells to be reprogrammed by the evolving tumor milieu into tumor-supportive macrophages that facilitate tumor progression and promote therapeutic evasion. In this study, we uncovered that macrophages from preneoplastic breast lesions were enriched for ribosome biosynthesis genes, indicating that this is an early event that is maintained in the tumor tissue. Furthermore, following treatment with irradiation or chemotherapy, breast tumors featured an abundance of tumor-supporting macrophages that displayed an enrichment of signatures of rRNA expression and ribosome biosynthesis. Consistently, rRNA synthesis was increased in tumor-supportive macrophages. In preclinical models of mammary cancer, a low dose of the RNA biogenesis inhibitor BMH-21 converted protumor macrophages to tumor-suppressive macrophages and supported an inflammatory tumor microenvironment. Inhibition of rRNA transcription stimulated a nucleolar stress response that activated the p53 and NF-κB pathways, which orchestrated impaired ribosome biogenesis checkpoint signaling that induced an inflammatory program in macrophages. Finally, inhibiting ribosome biogenesis augmented the effectiveness of neoadjuvant therapy. Together, these findings provide evidence that ribosome biogenesis is a targetable dependency to reprogram the tumor immune microenvironment. Significance: Increased ribosome biogenesis is an integral attribute of protumor macrophages that occurs early during breast tumorigenesis and represents a therapeutically actionable process to reactivate the tumor-suppressive functions of macrophages.

Atypical antipsychotics are widely used for the treatment of mental and behavioral disorders such as bipolar disorder, obsessive-compulsive disorder, and schizophrenia. However, these drugs can occasionally induce neutropenia or agranulocytosis, characterized by a significant reduction in circulating neutrophils, the primary white blood cells responsible for immune responses. This drug-induced neutropenia poses a considerable risk of life-threatening infections. However, the precise mechanism by which atypical antipsychotics induce neutropenia remains unclear. This study investigates the effects of four atypical antipsychotics, namely – aripiprazole, clozapine, olanzapine, and quetiapine – on the human neutrophil model cell line HL-60. These drugs, which modulate dopamine receptor signaling alongside other mechanisms, were analyzed for their effects. Among these, aripiprazole – but not the others – uniquely induced apoptosis in a dose-dependent manner, accompanied by an increased expression of pro-apoptotic genes – BAK, BCL10, and caspase-3. Moreover, our study elucidates that while differentiated HL-60 cells express D1-like and D2-like dopamine receptors, aripiprazole’s cytotoxic effects appear to operate through dopamine-independent pathways and significantly reduce phosphorylated Src family kinase levels. Our results align with previous studies suggesting that aripiprazole exhibits cytotoxic properties in neutrophils. Nevertheless, further investigations are warranted to investigate the mechanisms underlying aripiprazole-induced apoptosis in neutrophils.

Intratumoral low oxygen tension promotes cancer cell invasion and metastasis. Hypoxia-Inducible Factor 1-alpha (HIF1α) is the principal transcription factor orchestrating cellular responses to hypoxic stress, mediating the regulation of genes implicated in adapting to perturbations in oxygen homeostasis. Here, we describe our findings that functionally demonstrate a nucleolar localization domain in HIF1ɑ that enables HIF1ɑ to translocate to the nucleolus. Nucleolar HIF1ɑ binds the ribosomal DNA promoter and upregulates RNA Polymerase I activity leading to dysregulated ribosomal RNA transcription and consequently enhanced ribosome biogenesis. Ribosome biogenesis is important in supporting cellular metabolic processes and invasion and metastasis. Our findings are recapitulated in breast tumors wherein upregulated HIF1ɑ and rRNA biogenesis are associated with poor prognosis. Finally, our studies demonstrate that inhibition of RNA Polymerase I impedes aggressive traits of hypoxia-driven cancer progression, highlighting the potential of this approach as a therapeutic strategy in breast cancer. Cumulatively our work unravels an unprecedented role of HIF1ɑ in regulating rRNA biogenesis in breast cancer.