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T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4 + , and CD8 + memory T cells targeting the mutated KRAS G12D and KRAS G12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8 + neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients. Adoptive cell therapy (ACT) using neoantigen-specific T cells can lead to tumor regression. Here the authors use an in vitro stimulation approach to isolate tumor specific memory T cells from peripheral blood of metastatic epithelial cancer patients targeting unique as well as shared mutations in the KRAS oncogene.

Both environmental cues and intracellular bioenergetic states profoundly affect intraceUular pH （pHi）. How a cell responds to phi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein （BMP） signaling responds to pHi changes. Cold, basic or hypertonic conditions increase phi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of SmadS, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.

Abstract In teleosts, homologs of Anti-Müllerian Hormone (Amhy) and its type II receptor (Amhr2/Amhr2y) have been independently recruited as master sex-determination genes in about 50% of known cases. However, it remains unknown whether a conserved transducer pair exists, as the requisite type I receptors and R-Smad effectors remain unidentified amidst their diversity and potential redundancy. In this study, we employed an in vitro reporter assay to screen five type I receptors (Alk2a, Alk2b, Alk3, Alk6a, Alk6b) and three R-Smads (Smad1, Smad5, Smad8), discovering that only Alk3, Alk6a, or Alk6b, in combination with Smad5, significantly activated Amhy/Amhr2 signaling. In Nile tilapia, levels of phosphorylated Smad5 (p-Smad5) were notably elevated in XY gonads compared with XX gonads during the critical sex-determination window (8 to 15 dpf), while total Alk3 and Smad5 expression did not exhibit sexual dimorphism. The inhibition of type I receptors in XY fish resulted in feminization or complete sex reversal. Similarly, CRISPR/Cas9 mutagenesis of alk3 or smad5 led to male-to-female sex reversal in F0 mosaic mutants. Importantly, homozygous mutations in alk3 or smad5 resulted in embryonic lethality at the gastrula stage, whereas mutations in other type I receptors or R-Smads were viable and demonstrated normal sexual development. The conservation of this pathway was further substantiated in Southern catfish, where mutations in alk3a or smad5 also induced sex reversal in XY individuals. Collectively, our findings establish Alk3 and Smad5 as essential and specific transducers of the Amhy/Amhr2-mediated sex-determination pathway, revealing a potentially conserved signaling axis across teleosts.

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-β–SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells. Zhu et al. show how cancer-associated fibroblasts (CAFs) regulate metabolism of branched-chain amino acids in pancreatic ductal adenocarcinomas. CAFs secrete and deliver branched-chain ketoacids to cancer cells by degrading proteins in the extracellular matrix that are internalized from the tumour microenvironment.

SMAD5 has been demonstrated to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the circ_0001825/miR-1270/SMAD5 axis or KCNQ1OT1/miR-320a/SMAD5 axis. Therefore, SMAD5 may be a key regulator of BMSCs osteogenic differentiation, and its more related molecular mechanisms are worth further revealing. Western blot analysis was used to detect the protein levels of SMAD5, ubiquitin-specific peptidase 53 (USP53), eukaryotic translation initiation factor 4A3 (EIF4A3), and osteogenic differentiation-related markers. Cell counting kit 8 and transwell assay were performed to measure cell viability and invasion. Alkaline phosphatase (ALP) activity detection and Alizarin red S staining were employed to assess osteogenic differentiation. The interactions between USP53 and SMAD5/EIF4A3 were confirmed by Co-immunoprecipitation assay. The mRNA levels of SMAD5 and USP53 were examined using quantitative real-time PCR. SMAD5 silencing suppressed viability, invasion and osteogenic differentiation of BMSCs, while its overexpression had opposite effects. USP53 deubiquitinated SMAD5 to stabilize its protein expression. Moreover, USP53 knockdown inhibited viability, invasion and osteogenic differentiation of BMSCs, while these effects were reverted by SMAD5 overexpression. EIF4A3 stabilized USP53 mRNA expression, and the inhibitory effect of EIF4A3 silencing on viability, invasion and osteogenic differentiation of BMSCs was abolished by USP53 overexpression. Furthermore, EIF4A3 enhanced SMAD5 expression by interacting with USP53. EIF4A3-stabilized USP53 promotes SMAD5 deubiquitination to enhance viability, invasion and osteogenic differentiation of BMSCs.

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.

Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-β and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4 , with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature. The establishment of functional vasculatures requires the specification of newly formed vessels into veins and arteries. Here, Neal et al. use a combination of genetic approaches in mice and zebrafish to show that BMP signalling, via ALK3 and SMAD1/5, is required for venous specification during blood vessel development.

Marco Sandri, Helge Amthor, Stefano Piccolo and colleagues show that BMP signaling is a key positive regulator of muscle hypertrophy. They further show that inhibiting BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of myostatin knockout mice and exacerbates the effects of denervation and fasting. Cell size is determined by the balance between protein synthesis and degradation. This equilibrium is affected by hormones, nutrients, energy levels, mechanical stress and cytokines. Mutations that inactivate myostatin lead to excessive muscle growth in animals and humans, but the signals and pathways responsible for this hypertrophy remain largely unknown. Here we show that bone morphogenetic protein (BMP) signaling, acting through Smad1, Smad5 and Smad8 (Smad1/5/8), is the fundamental hypertrophic signal in mice. Inhibition of BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of myostatin-deficient mice and strongly exacerbates the effects of denervation and fasting. BMP-Smad1/5/8 signaling negatively regulates a gene ( Fbxo30 ) that encodes a ubiquitin ligase required for muscle loss, which we named muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1). Collectively, these data identify a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy.

The Warburg effect serves as a crucial aspect of tumor metabolism, where tumor cells preferentially rely on glycolysis, despite its lower efficiency, over oxidative phosphorylation for energy production even under aerobic conditions. This reprogramming of glucose metabolism confers glioma cells with the capacity for survival and proliferation. Serving as a messenger for regulating transforming growth factor beta, intracellular pH, cell metabolism maintaining cellular bioenergetic homeostasis, SMAD family member 5 (SMAD5) plays a pivotal role in the malignant progression of glioma cells and aerobic glycolysis. Hence, we have identified the expression and function of SMAD5 in human glioma cells, aiming to clarify its role in glycolysis. qRT-PCR and Western blot, reveal that SMAD5 is significantly overexpressed in glioma cells. Knocking down SMAD5 can effectively suppress the proliferation and invasion of glioma cells, while promoting apoptosis, furthermore, downregulation of SMAD5 in vivo has been shown to significantly reduce the growth of xenograft tumors. Conversely, overexpressing SMAD5 enhances the proliferative and invasive capabilities of glioma cells, while suppressing apoptosis. Concurrently, alterations in the expression level of SMAD5 exert an impact on the expression of glucose transporter GLUT1 and crucial enzymes involved in glycolysis, namely HK2 and PKM2, ultimately influencing the glycolytic capability of glioma cells. Specifically, knockdown of SMAD5 suppresses glycolysis, whereas its overexpression enhances glycolytic activity. In conclusion, our data demonstrate that SMAD5 can influence the proliferation, invasion, and apoptosis of glioma cells by modulating glycolysis. This finding holds potential for the development of novel metabolic treatment strategies for glioma.

The best characterized signaling pathway downstream of transforming growth factor β (TGF-β) is through SMAD2 and SMAD3. However, TGF-β also induces phosphorylation of SMAD1 and SMAD5, but the mechanism of this phosphorylation and its functional relevance is not known. Here, we show that TGF-β-induced SMAD1/5 phosphorylation requires members of two classes of type I receptor, TGFBR1 and ACVR1, and establish a new paradigm for receptor activation where TGFBR1 phosphorylates and activates ACVR1, which phosphorylates SMAD1/5. We demonstrate the biological significance of this pathway by showing that approximately a quarter of the TGF-β-induced transcriptome depends on SMAD1/5 signaling, with major early transcriptional targets being the ID genes. Finally, we show that TGF-β-induced epithelial-to-mesenchymal transition requires signaling via both the SMAD3 and SMAD1/5 pathways, with SMAD1/5 signaling being essential to induce ID1. Therefore, combinatorial signaling via both SMAD pathways is essential for the full TGF-β-induced transcriptional program and physiological responses. Cells communicate with other cells via signaling molecules to coordinate their activities. Signals released from one cell can influence the behavior of neighboring cells. Signaling molecules belonging to the TGF-β family play crucial roles in animals. For example, these molecules guide the formation of tissues and organs and help maintain them throughout the animal’s adult life. Abnormal regulation of TGF-β family signaling can fuel the growth of cancer cells and also contribute to other diseases in humans. Molecules in the TGF-β family bind to and bring together specific receptors on the surface of the receiving cell. This allows the receptors to activate so-called SMAD proteins within that cell. Activated SMADs move to the cell’s nucleus, where they regulate the activity of target genes. This in turn changes how the cell behaves. The best-studied member of the TGF-β family is TGF-β itself. It is well known to activate two particular SMAD proteins called SMAD2 and SMAD3. Recent research showed that TGF-β could also activate two different SMAD proteins, SMAD1 and SMAD5. However, it was not understood how this was achieved, or what its biological consequences were. Ramachandran et al. set out to address these questions in mouse and human cells grown in the laboratory. The experiments showed that, in addition to its known dedicated receptors, TGF-β also requires a third receptor to activate SMAD1 and SMAD5. Also, TGF-β signaling leads to changes in the activity of several thousand genes, and approximately a quarter of them require signaling via SMAD1 and SMAD5. Further work showed that SMAD1 and SMAD5 are needed for a process called epithelial-to-mesenchymal transition. This is a normal part of animal development, and is also a common feature of cancer cells, allowing them to spread to distant parts of the body. Understanding of how TGF-β signaling works in more detail may reveal new ways to target this pathway to treat diseases like cancer. The next step is to see how the signaling via SMAD1 and SMAD5 contributes to different aspects of cancer development.