Research
Abstract
Hypoxia critically restricts the effectiveness of immunotherapy in triple-negative breast cancer (TNBC). Comprehensive bioinformatics analyses demonstrated that highly hypoxic TNBC tumors exhibited elevated T cell exhaustion, increased immune checkpoint molecule expression, and diminished responsiveness to immune checkpoint blockade (ICB). Consequently, strategies aimed at alleviating tumor hypoxia may effectively augment ICB therapy. Although ultrasound-targeted microbubble cavitation (UTMC) has been shown to reduce tumor hypoxia, the precise molecular mechanisms remain unclear. Here, we provided evidence that UTMC activated endothelial nitric oxide synthase (eNOS) through G protein–coupled signaling, resembling pathways induced by fluid shear stress. UTMC-induced eNOS activation was largely Ca²⁺-dependent and resulted in increased nitric oxide production. Enhanced nitric oxide generation was associated with improved tumor perfusion and reduced hypoxia. Combining UTMC with anti–PD-L1 therapy markedly improved the tumor immune microenvironment, characterized by increased CD8+ T cell infiltration, reduced T cell exhaustion, diminished regulatory T cell infiltration, increased macrophage polarization from an M2 to M1 phenotype, and elevated production of pro-inflammatory cytokines. Collectively, our findings identified UTMC as a promising adjunctive therapeutic approach to mitigate hypoxia and enhance the efficacy of anti–PD-L1 immunotherapy in TNBC. These results support further translational evaluation of UTMC-based combination strategies in hypoxic TNBC.
Authors
Zhiyu Zhao, Li Ba, Siwei Li, Jianxin Wang, Yuzhou Luo, Sihan Wang, Yan Jin, Changjun Wu
Abstract
Pancreatic ductal adenocarcinoma (PDAC) shows profound resistance to immunotherapy due to its immunosuppressive tumor microenvironment. Here, we studied the relationship between T cell infiltration and innate immune signaling in PDAC, identifying Toll-like receptor 2 (TLR2) as a key regulator of T cell exclusion. TLR2 expression correlated with T cell infiltration in both human and mouse PDAC tumors. Using genetic knockout models and adoptive T cell transfer experiments, we found that TLR2 expression in both T cells and non-T cells contributes to T cell exclusion in PDAC. Notably, successful infiltration of adoptively transferred tumor-specific T cells required TLR2 deletion in both the transferred cells and the recipient host. The therapeutic implications of these findings are demonstrated through both genetic deletion and pharmacological inhibition of TLR2 using AAV-mediated and antibody-based approaches in murine models, resulting in decreased tumor growth and extended survival. Collectively, these findings identify TLR2 as a key modulator of T cell trafficking and immune suppression within the PDAC microenvironment, suggesting its potential as a therapeutic target for improving treatment outcomes.
Authors
Jacqueline Plesset, Meredith L. Stone, John C. McVey, Heather Coho, Kelly Markowitz, Kayjana Coho, Jesse Lee, Anna S. Thickens, Devora Delman, Gregory L. Beatty
Abstract
HIV infection rapidly impairs the gastrointestinal (GI) barrier, contributing to persistent mucosal immune dysfunction, microbial translocation, and systemic inflammation despite antiretroviral therapy (ART). Using SIV-infected rhesus macaques on long-term ART, we investigated mechanisms underlying impairment in gut barrier-protective IL-17/IL-22 responses and the potential modulation of this pathway by dietary indoles. Longitudinal profiling of colonic epithelial and lamina propria cells revealed a selective loss of IL-17/IL-22–producing γδT cells and type 3 innate lymphoid cells (ILC3s). This loss correlated with reduced expression of the transcription factors AHR and RORγt and was associated with elevated plasma markers of intestinal epithelial barrier disruption (IEBD), including intestinal fatty acid–binding protein (iFABP), zonulin, and LPS-binding protein (LBP). Targeting this transcriptional deficiency, dietary indole supplementation for one month restored colonic AHR⁺IL-22-producing γδ T cells, RORγt⁺ ILC3s, and Vδ1 T cells, and was associated with reduced iFABP and zonulin levels. Immunohistochemical analyses further demonstrated enrichment of AHR/RORγt-co-expressing cells in the colon of indole-supplemented animals during chronic SIV infection on ART. Collectively, these findings indicate that disruption of the AHR-RORγt axis is a key pathogenic mechanism underlying persistent IEBD in chronic SIV/HIV infection. Modulation of AHR and RORγt signaling pathways in the gut may therefore represent a promising therapeutic strategy to reinforce mucosal barrier function and mitigate chronic inflammation in people living with HIV.
Authors
Siva Thirugnanam, Alison R. Van Zandt, Alexandra B. McNally, Victoria A. Hart, Isabelle Berthelot, Cecily C. Midkiff, Lara A. Doyle-Meyers, David A. Welsh, Robert V. Blair, Andrew G. MacLean, Namita Rout
Abstract
Spinocerebellar Ataxia Type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in the gene encoding protein kinase C gamma (PKCγ), a Ca2+/diacylglycerol (DG)-dependent serine/threonine kinase dominantly expressed in cerebellar Purkinje cells. These mutations impair autoinhibitory constraints to increase the basal activity of the kinase, resulting in deficits in the cerebellum that are not observed upon simple deletion of the gene, and severe ataxia. To better understand the impact of aberrant PKCγ signaling in disease pathology, we developed a knock-in murine model of the SCA14 mutation ΔF48 in PKCγ. This fully-penetrant mutation is severe in humans and is mechanistically informative as it has high basal activity but is unresponsive to agonist stimulation. Genetic, behavioral, and molecular testing revealed that ΔF48 PKCγ mice have ataxia-related phenotypes and an altered cerebellar phosphoproteome driven primarily by enhanced Ca2+/calmodulin-dependent Kinase II (CaMKII) signaling, effects that were more severe in male mice. Analysis of existing human data revealed that SCA14 has a significantly earlier age of onset for males compared with females. Data from this clinically relevant mutation suggested that enhanced basal activity of PKCγ is sufficient to cause ataxia and that treatment strategies to modulate aberrant PKCγ may be particularly beneficial in males.
Authors
Sarah A. Wolfe, Yuliang Ma, Tomer M. Yaron-Barir, Carly Chang, Caila A. Pilo, Majid Ghassemian, Amanda J. Roberts, Sang Ryeul Lee, Benjamin A. Henson, Kristen Jepsen, Jared L. Johnson, Lewis C. Cantley, Susan S. Taylor, George Gorrie, Alexandra C. Newton
Abstract
Huntington’s disease (HD) is a fatal neurodegenerative disease caused by an expanded polyglutamine (CAG) repeat in the N-terminal of the Huntingtin protein (HTT). Microglial activation and elevated pro-inflammatory cytokines are observed in HD brains, but the mechanisms regulating neuroinflammation and microglial activation are poorly understood. Metformin-mediated neuroprotection has been demonstrated in experimental models of neurodegeneration, including HD. We found that metformin inhibits mitochondrial DNA (mtDNA) release and subsequent neuroinflammation in the cortex and striatum of a mouse model of HD. Moreover, elevated pro-inflammatory cytokines and microglial activation are inhibited by metformin in HD transgenic mice brain. Metformin reduced pathological microglial clusters and shifted towards a quiescent, homeostatic phenotype. Metformin improved aberrant immunometabolism in HD mouse brain and primary microglia. Mechanistically found that metformin regulates mitochondrial fission, reprograms deregulated metabolism in HD microglia, and controls microglial activation and inflammation in HD transgenic mice.
Authors
Abhishek Jauhari, Adam C. Monek, Olena S. Abakumova, Tanisha Singh, Sukhman Singh, Xiaomin Wang, Carley S. Clise, Diane L. Carlisle, Robert M. Friedlander
Abstract
The RhoBTB1-Cullin3 (CUL3) pathway in smooth muscle cells (SMCs) controls the ubiquitination and proteasomal degradation of target proteins that regulate vasodilation, vasoconstriction, and the actin cytoskeleton, and through this blood pressure (BP) and arterial stiffness. Using proximity labelling coupled with mass spectrometry in A7R5 SMCs, we identified proteins which bound to the C-terminal half of RhoBTB1 which functions as an adapter to deliver substrates to CUL3. We examined the physiological relevance of one of these substrates, RbFox2. Co-immunoprecipitation validated the interaction of RbFox2 with RhoBTB1. RbFox2 expression was elevated in response to inhibition of the ubiquitination-proteasomal pathway, CUL3-deficiency, and RhoBTB1 inhibition by either siRNA or angiotensin II (ANG). RbFox2 was ubiquitinated in a RhoBTB1- and CUL3-dependent manner suggesting its regulation through the RhoBTB1-CUL3-dependent ubiquitin-proteasome pathway. Inhibition of RbFox2 impaired the actin cytoskeleton in A7R5 cells and in primary SMC from RbFox2Flox/Flox (RbFox2F/F) mice and decreased the levels of globular and filamentous actin. ANG increased BP and arterial stiffness of RbFox2F/F mice, but the progression of arterial stiffness was halted after SMC-specific RbFox2 deletion despite a continued rise in BP. We conclude that RhoBTB1 and RbFox2 are important regulators of arterial stiffness through a mechanism that influences cytoskeletal integrity.
Authors
Gaurav Kumar, Nisita Chaihongsa, Daniel T. Brozoski, Daria Golosova, Ibrahim Vazirabad, Ko-Ting Lu, Kelsey K. Wackman, Ravi K. Singh, Curt D. Sigmund
Abstract
VIC-1911 (formerly TAS-119) is a next-generation, ATP-competitive Aurora kinase A (AURKA) inhibitor with a favorable biosafety profile. However, it has not been evaluated in prostate cancer (PC), wherein AURKA is highly expressed in advanced stages and represents a critical therapeutic target. Here, we demonstrate that VIC-1911 potently inhibits AURKA activity with high selectivity over AURKB/C across diverse PC cell lines. Treatment with VIC-1911, even at nanomolar concentrations, substantially inhibits the growth of both androgen receptor (AR)-positive and AR-negative PC cells. VIC-1911 triggers mitotic failure, induces DNA double-strand breaks (DSBs), and activates the p53 pathway, halting cell division and inducing cell death. Notably, VIC-1911 showed synergistic effects in inhibiting PC cell growth in vitro and xenograft tumor growth in vivo with poly (ADP-ribose) polymerase inhibitors (PARPi), which have proven effective in PC with a deficiency in Homologous Recombination (HR) repair. Mechanistically, VIC-1911 disabled HR-mediated repair of DSBs in otherwise HR-proficient PC cells, leading to a “BRCAness” phenotype and pronounced accumulation of DNA damage and mitotic catastrophe. In summary, our study uncovers what we believe a novel mechanism to functional “BRCAness” by inducing mitotic arrest and highlights VIC-1911 as a promising therapeutic agent for advanced PC, either as a single agent or in combination, sensitizing HR-proficient tumors to PARP inhibitors.
Authors
Galina Gritsina, Sandip Kumar Rath, Hongshun Shi, Qi Chu, Wanqing Xie, Que Thanh Thanh Nguyen, Sambhavi Senthil, Thomas J. Myers, Mehmet A. Bilen, Sarah E. Fenton, Maha Hussain, David S. Yu, Jonathan C. Zhao, Jindan Yu
Abstract
β-arrestins are ubiquitously expressed cytosolic adaptor proteins that regulate G protein-coupled receptor-dependent and -independent pathways essential for numerous physiological functions. This study investigated the role of β-arrestin1 and -2 in embryonic lymphatic vessel development and survival by generating and characterizing mice with lymphatic, tamoxifen-inducible loss of the genes encoding β-arrestin-1 and -2 (Arrb1/2ΔiLEC). At embryonic day15.5 (E15.5), Arrb1/2ΔiLEC embryos exhibit profound hydrops fetalis and increased embryonic mortality compared to control Arrb1/2fl/fl embryos. Edematous Arrb1/2ΔiLEC embryos, which were more often represented by the female sex, showed growth restriction and decreased lymphatic endothelial cell (LEC) proliferation in the jugular lymphatic sac compared to controls. In vitro knockdown of β-arrestin1 in LECs increased proliferation and increased activation of AKT, while knockdown of β-arrestin2 decreased proliferation and decreased activation of both ERK and CREB. Arrb1/2ΔiLEC embryos also exhibited dilated dermal lymphatics with decreased continuous VE-Cadherin adherens junctions compared to controls. These results were recapitulated in vitro in β-arrestin1 and/or -2 knockdown human LECs, which showed a decrease in membrane VE-Cadherin and β-catenin levels, and prevention of adrenomedullin-induced linearization of VE-cadherin at endothelial cell–cell junctions. Collectively, these results demonstrate that loss of β-arrestin1/2 in lymphatics causes hydrops fetalis, mid-gestational growth arrest and embryonic demise associated with reduced LEC proliferation and disrupted VE-Cadherin adherens junctions.
Authors
Yanna Tian, D. Stephen Serafin, Monserrat Avila-Zozaya, Alyssa M. Tauro, Natalie M. Torres-Valle, Bryan M. Kistner, Danielle M. Dy, Elizabeth S. Douglas, Kathleen M. Caron
Abstract
Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene leading to decreased SMN protein levels and motor neuron dysfunction. SMN-restoring therapies offer clinical benefit, but the downstream molecular consequences of SMN reduction remain incompletely understood. SMN deficiency resulted in downregulation of kinesin heavy chain isoform 5A (KIF5A) in human neurons and in a mouse model of SMA. SMN associated with KIF5A mRNA and contributed to its stability. Reduced SMN levels impaired axon regeneration, which was rescued by KIF5A overexpression. Because KIF5A has also been connected to ALS, these findings provide evidence of a molecular link between SMA and ALS pathophysiology, highlighting KIF5A as an SMN regulated factor. Our findings suggest SMN-independent interventions targeting KIF5A could represent a complementary therapeutic approach for SMA and other motor neuron diseases.
Authors
Tetsuya Akiyama, Yi Zeng, Caiwei Guo, Olivia Gautier, Lauren Koepke, Heankel Lyons, Elana Molotsky, Juliane S. Bombosch, Odilia Sianto, Jay P. Ross, Phuong Hoang, Luke Zhao, Cole Spencer, Charlotte J. Sumner, Michelle Monje, John W. Day, Aaron D. Gitler
Abstract
Thoracic Aortic Aneurysm and Dissections (TAAD) is a progressive dilation of the aortic wall associated with degradation of the extracellular matrix (ECM), cystic medial degeneration, smooth muscle cell (SMC) dysfunction, and rarefaction. TAAD etiology and pathogenesis suggest that alteration of mechanical force propagation may contribute to SMC dysfunction. This study aims to determine the role of SMC focal adhesion proteins, which are key components of force transmission, in TAAD pathogenesis. scRNAseq analysis of human TAA aortas showed reduced expression of intracellular focal adhesion components, including PTK2 (FAK), VCL, ILK, and TES transcripts, in SMCs. Additionally, protein levels of FAK, ILK, and VCL were decreased in the aorta of patients with TAA. SMC-specific Ptk2, Vcl, and Ilk knockout mice treated with β-Aminopropionitrile (BAPN) exhibited increased mortality, aortic dilation, ECM breakdown, and SMC loss. Mechanistically, knocking down FAK, ILK, and VCL exacerbated gliotoxin-induced SMC anoikis, whereas overexpressing full-length wild-type (WT) and dead-kinase FAK conferred resistance to apoptosis and cell detachment, indicating that FAK's protective effects depend on its expression rather than its enzymatic activity. Inhibition of FAK kinase activity did not affect SMC apoptosis in vitro or aortic dilation in vivo. Our findings demonstrated that the expression of focal adhesion proteins protects against TAAD progression and SMC anoikis independently of FAK kinase activity.
Authors
Zhenyuan Zhu, Mingjun Liu, Jianxin Wei, Deepa Suryanarayan, Parya Behzadi, Robert Edgar, Julie A. Phillippi, Cynthia St. Hilaire, Cristina Espinosa-Diez, Delphine Gomez
No posts were found with this tag.
