Cell biology
Abstract
Sarcomas are a heterogeneous group of cancers with few shared therapeutic targets. We show that PI3K signaling is frequently activated in sarcomas due to PTEN loss (in 30-60%), representing a common therapeutic target. The PI3K pathway has lacked a downstream oncogenic transcription factor. We show TAZ and YAP are transcriptional co-activators regulated by PI3K and drive a transcriptome necessary for tumor growth in a PI3K-driven sarcoma mouse model. This PI3K-TAZ/YAP axis exists in parallel to the known PI3K-Akt-mTORC1 axis providing a rationale for combination therapy targeting the TAZ/YAP-TEAD interaction and mTORC1. Combination therapy using IK-930 (TEAD inhibitor) and everolimus (mTORC1 inhibitor) synergistically diminished proliferation and anchorage dependent growth of PI3K-activated sarcoma cell lines at low, physiologically achievable doses. Furthermore, this combination therapy showed a synergistic effect in vivo, suggesting that an integrated view of PI3K and Hippo signaling can be leveraged therapeutically in PI3K activated sarcomas.
Authors
Keith C. Garcia, Ali A. Khan, Krishnendu Ghosh, Souradip Sinha, Nicholas Scalora, Gillian DeWane, Colleen Fullenkamp, Nicole Merritt, Yuliia Drebot, Samuel Y. Yu, Mariah Leidinger, Michael D. Henry, Patrick J. Breheny, Michael S. Chimenti, Munir R. Tanas
Abstract
Adaptive remodeling of retrodiscal tissue following anterior disc displacement (ADD) of the temporomandibular joint (TMJ) has been recognized for decades, yet the underlying cellular dynamics and molecular mechanisms remain unclear. Using a porcine ADD model, this study investigated the cellular and molecular basis driving retrodiscal tissue adaptation. Histological staining revealed adaptive remodeling of retrodiscal tissue after ADD induction, with dense connective tissue and cartilaginous masses replacing loose connective tissue. Furthermore, single-cell RNA sequencing (scRNA-seq) captured pronounced fibroblast expansion during tissue remodeling, notably the FB2 subcluster with high developmental potential, and the emergence of a mural cell subcluster MC4 associated with extracellular matrix (ECM) remodeling. CellChat analysis highlighted MC4-FB2 crosstalk via FGF2 and BMP5 signaling. The combination of pathway-aware multi-layered hierarchical network (P-NET) and Seurat with drug database screening identified five promising compounds. Among them, Zaprinast demonstrated the most robust effects by enhancing the remodeling capability of fibroblasts in vitro, and also alleviated TMJ deformation in vivo. Collectively, fibroblast activation is pivotal for early retrodiscal tissue adaptation following ADD, which is driven by MC4-derived FGF2/BMP5 signaling. Zaprinast treatment potentiates this remodeling process. These findings provide new insights into cellular basis of TMJ adaptation and identify potential therapeutic targets for ADD management.
Authors
Wenlin Yuan, Yilin Chen, Ruojin Yan, Wei Liu, Chenyu Wang, Ying Wang, Qiaoli Dai, Wen Li, Mengqi Zhu, Xiao Chen, Jiejun Shi
Abstract
The chronic inflammation of Crohn’s disease frequently leads to fibrosis and muscular hypertrophy of the intestinal wall. This often culminates in strictures, a serious condition lacking directed therapy. Severe pathological changes occur in the submucosa and muscularis propria intestinal wall layers of strictures, yet stricture-associated proteome changes in these layers is unexplored. We perform unbiased proteomics on submucosa and muscularis propria microdissected from transmural sections of strictured and non-strictured ileum. Proteome changes in stricture submucosa reflect a transition from homeostasis to tissue remodeling, inflammation and smooth muscle alterations. Top submucosa features include reduced vascular components and lipid metabolism proteins accompanied by increased proteins with immune-, matrix- or stress functions including CTHRC1, TNC, IL16, MZB1 and TXNDC5. In parallel, predominant changes in stricture muscularis propria include increased matrix (POSTN) and immune (mast cell CPA3) proteins alongside decreased proteins with lipid metabolic, mitochondrial or key muscle functions. Finally, trends of differentially expressed proteins along non-stricture submucosa suggest progressive profibrotic tissue remodeling and muscle expansion as proximity to stricture increases. The comprehensive proteome map presented here offers unique layer-resolved insight into the stricture microenvironment and potential drivers of fibrotic disease, providing a valuable resource to fuel biomarker and therapeutic target research. Keywords: Crohn’s disease strictures, proteomics, intestinal fibrosis, fibrostenosis
Authors
Johannes Alfredsson, Carina Sihlbom Wallem, Maja Östling, Hanna de la Croix, Elinor Bexe-Lindskog, Mary Jo Wick
Abstract
In pemphigus, autoantibodies against the desmosomal cadherins desmoglein (DSG) DSG1 and DSG3 cause intraepidermal blistering. Recently, we found that increasing cAMP with the phosphodiesterase-4 inhibitor apremilast stabilizes keratinocyte cohesion in pemphigus. This effect is paralleled by phosphorylation of the desmosomal plaque protein plakoglobin (PG) at serine 665 (S665). Here, we investigated the relevance of PG phosphorylation at S665 for stabilization of keratinocyte cohesion and further characterized the underlying mechanisms. Ultrastructural analysis of a recently established PG-S665 phospho-deficient mouse model (PG-S665A) showed diminished keratin insertion. Accordingly, the protective effect of apremilast against pemphigus autoantibody-induced skin blistering was diminished, and apremilast failed to restore alterations of the keratin cytoskeleton in PG-S665A mice. Keratinocytes derived from PG-S665A mice revealed a disorganized keratin cytoskeleton and reduced single-molecule binding strength of DSG3. In line with this, in ex vivo human skin, increased cAMP augmented keratin insertion into desmosomal plaques. Additionally, PG phosphorylated at S665 colocalized with desmoplakin and keratin filaments anchoring to desmosomes and increased cAMP-accelerated assembly of desmosomes. Taken together, phosphorylation of PG at S665 was crucial for protective effects of apremilast in pemphigus and for maintenance of DSG3 binding and keratin filament anchorage to desmosomes.
Authors
Franziska Vielmuth, Anna M. Sigmund, Desalegn T. Egu, Matthias Hiermaier, Letyfee S. Steinert, Sina Moztarzadeh, Mariia Klimkina, Margarethe E.C. Schikora, Paulina M. Rion, Thomas Schmitt, Katharina Meier, Kamran Ghoreschi, Anja K.E. Horn, Mariya Y. Radeva, Daniela Kugelmann, Jens Waschke
Abstract
Mutations in LMNA, encoding nuclear lamina protein Lamin A/C, cause premature aging disorders, most notably Hutchinson-Gilford Progeria Syndrome. Despite obvious skull abnormalities in progeroid patients, the disease-causing mechanism remains elusive. The L648R single amino acid substitution blocks prelamin A maturation in mice, modeling a unique human patient. Here, we describe skull deformities in premature aging caused by aberrant suture fusion resembling those of patients with craniosynostosis. Further examinations identify prelamin A accumulation causatively linked to multiple suture synostoses in low bone density. This etiology is distinct from conventional suture fusion mediated by excessive ossification. In addition, the mutation disrupts skeletal stem cell stemness and subsequent stem cell-mediated proliferation and differentiation in osteogenesis. Intrasutural bones present in progeroid patients are highly reminiscent of synostosis caused by stem cell exhaustion. Comparative gene expression profiling further reveals cytoskeletal dynamics associated with skeletogenic cell aging and suture patency in mice and humans. Functional studies demonstrate that abnormal structures of progeric nuclei caused by prelamin A accumulation affect cytoskeleton organization and nucleoskeleton assembly essential for craniofacial skeletogenesis. Pharmacogenetic analyses indicate alleviation of osteogenic defects via actin polymerization. Our findings provide compelling evidence for nuclear and cytoskeletal defects, mediating stem cell-associated osteogenic deformities in progeroid disorders.
Authors
Kai Li, Trunee Hsu, Hitoshi Uchida, Tingxi Wu, Susan Michaelis, Howard J. Worman, Wei Hsu
Abstract
The survival of patients with acute myelogenous leukemia (AML) carrying mutations in TP53 is dismal. We report the results of a detailed characterization of responses to treatment ex vivo with the MDM2 inhibitor MI219, a p53 protein stabilizer, in AML blasts from 165 patients focusing analyses on TP53 wildtype (WT) patients. In total 33% of AML were absolute resistant to MDM2 inhibitor induced apoptosis, of which 45% carried TP53 mutation and 55% were TP53 WT. We conducted array-based expression profiling of ten resistant and ten sensitive AML cases with WT TP53 status, respectively, at baseline and after 2h and 6h of MDM2 inhibitor treatment. While sensitive cases showed the induction of classical TP53 response genes, this was absent or attenuated in resistant cases. In addition, the sensitive and resistant AML samples at baseline profoundly differed in the expression of inflammation-related and mitochondrial genes. No TP53 mutated AML patient survived. The 4-year survival of AML with defective MDM2 inhibitor induced TP53-mediated apoptosis despite WT TP53 was dismal at 19% when NPM1 was co-mutated and 6% when NPM1 was WT. In summary, we identified prevalent multi-causal defects in TP53-mediated apoptosis in AML resulting in extremely poor patient survival.
Authors
Josephine Dubois, Anthony Palmer, Darren King, Mohamed Rizk, Karan Bedi, Kerby A. Shedden, Sami N. Malek
Abstract
Psoriasis is a chronic inflammatory dermatosis characterized by pathological keratinocyte hyperproliferation and dysregulated immune activation. While ubiquitin-specific peptidase 16 (USP16) has been implicated in modulating multiple cellular signaling pathways, its functional role in psoriatic pathogenesis remains poorly understood. Our investigation revealed pronounced upregulation of USP16 expression in psoriatic epidermis compared to normal controls. Keratinocyte-specific USP16 knockdown demonstrated remarkable therapeutic efficacy, significantly ameliorating characteristic psoriatic phenotypes including epidermal hyperplasia and inflammatory infiltration. RNA sequencing analysis showed that USP16 has substantial effects on cell cycle transition and keratinocytes proliferation. Through KEGG analysis, it was found that USP16 primarily regulates the NLRP3 signaling pathway, leading to enhanced cell proliferation and inflammation. Mechanically, USP16 directly binds to the NLRP3 protein to eliminate K48 ubiquitination modification, enhancing the stability of the NLRP3 protein, activating inflammasome activity. Further studies showed that the therapeutic effect of reducing USP16 on psoriasis progression were counteracted by an NLRP3 activator and keratinocyte-specific NLRP3 overexpression adenovirus. Collectively, these results shed light on how USP16 promotes NLRP3 signaling in keratinocytes, exacerbating psoriasis development. This positive regulation highlights the potential of USP16 as a therapeutic target for psoriasis.
Authors
Nan Wang, Fangqian Guan, Yifan Lin, Bohao Sun, Jindan Dai, Xiejun Xu, Weibo Tang, Yanhua Ren, Xuliang Huang, Wenjie Gao, Xixi Chen, Litai Jin, Weitao Cong, Zhongxin Zhu
Abstract
We provide evidence that human and murine SLFN5 proteins are modulators of Type I IFN responses and the immune response in pancreatic cancer. Blocking expression of Slfn5 in PDAC enhances IFN-responses, suppresses tumor growth, and prolongs survival in immunocompetent mice. Notably, immunophenotypic analysis reveals a reduction in tumor-associated macrophages (TAMs) alongside an increase in tumor infiltrating effector cells in tumors over time. These findings implicate SLFN5 acts as an intracellular immune checkpoint and identify it as a unique therapeutic target for the development of therapies for PDAC and possibly other malignancies.
Authors
Mariafausta Fischietti, Markella Zannikou, Elspeth M. Beauchamp, Diana Saleiro, Aneta H. Baran, Briana N. Hryhorysak, Jamie N. Guillen Magaña, Emely Lopez Fajardo, Gavin T. Blyth, Brandyn A. Castro, Jason M. Miska, Catalina Lee-Chang, Priyam Patel, Elizabeth T. Bartom, Masha Kocherginsky, Frank Eckerdt, Leonidas C. Platanias
Abstract
Cachexia is a debilitating syndrome characterized by progressive skeletal muscle wasting, commonly affecting cancer patients, particularly those with pancreatic cancer. Despite its clinical significance, the molecular mechanisms underlying cancer cachexia remain poorly understood. In this study, we utilized single-nucleus RNA sequencing (snRNA-seq) and bulk RNA-seq, complemented by biochemical and histological analyses, to investigate molecular alterations in the skeletal muscle of the KPC mouse model of pancreatic cancer cachexia. Our findings demonstrated that KPC tumor growth induced myofiber-specific changes in the expression of genes involved in proteolytic pathways, mitochondrial biogenesis, and angiogenesis. Notably, tumor progression enhanced the activity of specific transcription factors that regulate the mTORC1 signaling pathway, along with genes involved in translational initiation and ribosome biogenesis. Skeletal muscle-specific, inducible inhibition of mTORC1 activity further exacerbated muscle loss in tumor-bearing mice, highlighting its protective role in maintaining muscle mass. Additionally, we uncovered new intercellular signaling networks within the skeletal muscle microenvironment during pancreatic cancer-induced cachexia. Together, our study revealed previously unrecognized molecular mechanisms that regulates skeletal muscle homeostasis and identified potential therapeutic targets for the treatment of pancreatic cancer–associated cachexia.
Authors
Bowen Xu, Aniket S. Joshi, Meiricris Tomaz da Silva, Silin Liu, Ashok Kumar
Abstract
The lymphatic system maintains fluid homeostasis and orchestrates immune cell trafficking throughout tissues. While extensively studied in cancer and lymphedema, its role in non-lymphoid organs, particularly the kidney, remains an emerging area of investigation. Previous research established molecular connections between NF-κB, VEGFR-3, and PROX-1 in regulating lymphatic growth during inflammation, and studies using global knockout mice revealed that the NF-κB1 subunit (p50) influences lymphatic vessel density. However, the role of RelA—a key component of the canonical NF-κB heterodimer—in regulating lymphatic growth and kidney function following acute kidney injury (AKI) remains unexplored. Using an inducible, predominantly lymphatic-specific RelA knockout mouse model, we demonstrate that RelA expression in VEGFR-3+ cells is essential for VEGFR-3 driven lymphangiogenesis following AKI. Knockout mice exhibited significantly worse kidney function, altered histological features, impaired VEGFR-3-dependent lymphangiogenesis, and dysregulated immune cell trafficking. Compensatory upregulation of PROX-1 and podoplanin occurred despite decreased VEGFR-3 and LYVE-1 total protein expression, suggesting complex regulatory mechanisms. Our findings suggest that RelA is a critical sensor for inflammation and regulator of protective lymphangiogenesis following kidney injury and provide insights into potential therapeutic targets for improved kidney injury outcomes.
Authors
Arin L. Melkonian, Amie M. Traylor, Anna A. Zmijewska, Kyle H. Moore, Gelare Ghajar-Rahimi, Stephanie Esman, Yanlin Jiang, Hani Jang, Babak J. Mehrara, Timmy C. Lee, James F. George, Anupam Agarwal
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