Comprehensive analysis of germline drivers in endometrial cancer.

BACKGROUND: We sought to determine the prevalence of germline pathogenic variants (gPVs) in unselected patients with endometrial cancer (EC), define biallelic gPVs within tumors, and describe their associations with clinicopathologic features. METHODS: Germline assessment of at least 76 cancer predisposition genes was performed in patients with EC undergoing clinical tumor-normal Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing from January 1, 2015, to June 30, 2021. In patients with gPVs, biallelic alterations in ECs were identified through analysis of loss of heterozygosity and somatic PVs. Clinicopathologic variables were compared using nonparametric tests. RESULTS: Of 1625 patients with EC, 216 (13%) had gPVs, and 15 patients had 2 gPVs. There were 231 gPVs in 35 genes (75 [32%] high penetrance; 39 [17%] moderate penetrance; and 117 [51%] low, recessive, or uncertain penetrance). Compared with those without gPVs, patients with gPVs were younger (P = .002), more often White (P = .009), and less obese (P = .025) and had differences in distribution of tumor histology (P = .017) and molecular subtype (P < .001). Among 231 gPVs, 74 (32%) exhibited biallelic inactivation within tumors. For high-penetrance gPVs, 63% (47 of 75) of ECs had biallelic alterations, primarily affecting mismatch repair (MMR) and homologous recombination related genes, including BRCA1,BRCA2, RAD51D, and PALB2. Biallelic inactivation varied across molecular subtypes with highest rates in microsatellite instability-high (MSI-H) or copy-number (CN)-high subtypes (3 of 12 [25%] POLE, 30 of 77 [39%] MSI-H, 27 of 60 [45%] CN-high, 9 of 57 [16%] CN-low; P < .001). CONCLUSIONS: Of unselected patients with EC, 13% had gPVs, with 63% of gPVs in high-penetrance genes (MMR and homologous recombination) exhibiting biallelic inactivation, potentially driving cancer development. This supports germline assessment in EC given implications for treatment and cancer prevention.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Recurrent WWTR1 S89W mutations and Hippo pathway deregulation in clear cell carcinomas of the cervix.

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Single-cell gene profiling and lineage tracing analyses revealed novel mechanisms of endothelial repair by progenitors.

Stem/progenitor cells (SPCs) have been implicated to participate in vascular repair. However, the exact role of SPCs in endothelial repair of large vessels still remains controversial. This study aimed to delineate the cellular heterogeneity and possible functional role of endogenous vascular SPCs in large vessels. Using single-cell RNA-sequencing (scRNA-seq) and genetic lineage tracing mouse models, we uncovered the cellular heterogeneity of SPCs, i.e., c-Kit+ cells in the mouse aorta, and found that endogenous c-Kit+ cells acquire endothelial cell fate in the aorta under both physiological and pathological conditions. While c-Kit+ cells contribute to aortic endothelial turnover in the atheroprone regions during homeostasis, recipient c-Kit+ cells of nonbone marrow source replace both luminal and microvessel endothelial cells in transplant arteriosclerosis. Single-cell pseudotime analysis of scRNA-seq data and in vitro cell experiments suggest that vascular SPCs display endothelial differentiation potential and undergo metabolic reprogramming during cell differentiation, in which AKT/mTOR-dependent glycolysis is critical for endothelial gene expression. These findings demonstrate a critical role for c-Kit lineage cells in aortic endothelial turnover and replacement, and may provide insights into therapeutic strategies for vascular diseases.

Single-Cell RNA-Sequencing and Metabolomics Analyses Reveal the Contribution of Perivascular Adipose Tissue Stem Cells to Vascular Remodeling.

OBJECTIVE: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-ß1 (transforming growth factor ß1) and transfection of microRNA (miR)-378a-3p mimics induced a similar metabolic reprogramming of PV-ADSCs, including upregulated mitochondrial potential and altered lipid levels, such as increased cholesterol and promoted smooth muscle differentiation. CONCLUSIONS: Single-cell RNA-sequencing allows direct visualization of PV-ADSC heterogeneity at a single-cell level and uncovers 2 subpopulations with distinct signature genes and signaling pathways. The function of PVAT in vascular regeneration is partly attributed to PV-ADSCs and their differentiation towards smooth muscle lineage. Mechanistic study presents miR-378a-3p which is a potent regulator of metabolic reprogramming as a potential therapeutic target for vascular regeneration.

Binding of Dickkopf-3 to CXCR7 Enhances Vascular Progenitor Cell Migration and Degradable Graft Regeneration.

RATIONALE: Vascular progenitor cells play key roles in physiological and pathological vascular remodeling-a process that is crucial for the regeneration of acellular biodegradable scaffolds engineered as vital strategies against the limited availability of healthy autologous vessels for bypass grafting. Therefore, understanding the mechanisms driving vascular progenitor cells recruitment and differentiation could help the development of new strategies to improve tissue-engineered vessel grafts and design drug-targeted therapy for vessel regeneration. OBJECTIVE: In this study, we sought to investigate the role of Dkk3 (dickkopf-3), recently identified as a cytokine promotor of endothelial repair and smooth muscle cell differentiation, on vascular progenitor cells cell migration and vascular regeneration and to identify its functional receptor that remains unknown. METHODS AND RESULTS: Vascular stem/progenitor cells were isolated from murine aortic adventitia and selected for the Sca-1 (stem cell antigen-1) marker. Dkk3 induced the chemotaxis of Sca-1+ cells in vitro in transwell and wound healing assays and ex vivo in the aortic ring assay. Functional studies to identify Dkk3 receptor revealed that overexpression or knockdown of chemokine receptor CXCR7 (C-X-C chemokine receptor type 7) in Sca-1+ cells resulted in alterations in cell migration. Coimmunoprecipitation experiments using Sca-1+ cell extracts treated with Dkk3 showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high-affinity Dkk3-CXCR7 binding with a dissociation constant of 14.14 nmol/L. Binding of CXCR7 by Dkk3 triggered the subsequent activation of ERK1/2 (extracellular signal-regulated kinases 1/2)-, PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)-, Rac1 (Ras-related C3 botulinum toxin substrate 1)-, and RhoA (Ras homolog gene family, member A)-signaling pathways involved in Sca-1+ cell migration. Tissue-engineered vessel grafts were fabricated with or without Dkk3 and implanted to replace the rat abdominal aorta. Dkk3-loaded tissue-engineered vessel grafts showed efficient endothelization and recruitment of vascular progenitor cells, which had acquired characteristics of mature smooth muscle cells. CXCR7 blocking using specific antibodies in this vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus compromising vascular remodeling. CONCLUSIONS: We provide a novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced vascular progenitor migration in vitro and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.

Role of Resident Stem Cells in Vessel Formation and Arteriosclerosis.

Vascular, resident stem cells are present in all 3 layers of the vessel wall; they play a role in vascular formation under physiological conditions and in remodeling in pathological situations. Throughout development and adult early life, resident stem cells participate in vessel formation through vasculogenesis and angiogenesis. In adults, the vascular stem cells are mostly quiescent in their niches but can be activated in response to injury and participate in endothelial repair and smooth muscle cell accumulation to form neointima. However, delineation of the characteristics and of the migration and differentiation behaviors of these stem cells is an area of ongoing investigation. A set of genetic mouse models for cell lineage tracing has been developed to specifically address the nature of these cells and both migration and differentiation processes during physiological angiogenesis and in vascular diseases. This review summarizes the current knowledge on resident stem cells, which has become more defined and refined in vascular biology research, thus contributing to the development of new potential therapeutic strategies to promote endothelial regeneration and ameliorate vascular disease development.

Smooth muscle cells differentiated from mesenchymal stem cells are regulated by microRNAs and suitable for vascular tissue grafts.

Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-ß1 (TGFß1), human umbilical cord-derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFß1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.

Adventitial Sca1+ Cells Transduced With ETV2 Are Committed to the Endothelial Fate and Improve Vascular Remodeling After Injury.

OBJECTIVE: Vascular adventitial Sca1+ (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1+ cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease. APPROACH AND RESULTS: We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1+ cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1+ adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin, Flk-1, and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regulations, by decreasing the expression of DNA-modifying enzymes ten-eleven translocation dioxygenases. Adventitial Sca1+ cells grafted on the adventitial side of wire-injured femoral arteries increased vascular wall hyperplasia compared with control arteries with no grafted cells. Arteries seeded with ETV2-transduced cells, on the contrary, showed reduced hyperplasia compared with control. CONCLUSIONS: These data give evidence that the genetic manipulation of vascular progenitors is a promising approach to improve vascular function after endothelial injury.