Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state.

Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFß), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)-a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.

Exosomal-miR-522-3p derived from cancer-associated fibroblasts accelerates tumor metastasis and angiogenesis via repression bone morphogenetic protein 5 in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a gastrointestinal tract malignancy. Exosomes secreted by cancer-associated fibroblasts (CAFs) are reported to participate in tumor progression by delivering noncoding RNA or small proteins. However, the function of exosomal miR-522-3p in CRC remains unclear. METHODS: CAFs were derived from tumor tissues, and exosomes were identified by western blot or TEM/NTA and originated from CAFs/NFs. The viability, invasion, and migration of HUVECs and CRC cells was examined using MTT, Transwell, and wound healing assays, respectively. The molecular interactions were validated using dual luciferase reporter assay and RIP. Xenograft and lung metastasis mouse models were generated to assess tumor growth and metastasis. RESULTS: Exosomes extracted from CAFs/NFs showed high expression of CD63, CD81, and TSG101. CAF-derived exosomes significantly increased the viability, angiogenesis, invasion, and migration of HUVECs and CRC cells, thereby aggravating tumor growth, invasion, and angiogenesis in vivo. miR-522-3p was upregulated in CAF-derived exosomes and CRC tissues. Depletion of miR-522-3p reversed the effect of exosomes derived from CAFs in migration, angiogenesis, and invasion of HUVECs and CRC cells. Furthermore, bone morphogenetic protein 5 (BMP5) was identified as a target gene of miR-522-3p, and upregulation of BMP5 reversed the promoting effect of miR-522-3p mimics or CAF-derived exosomes on cell invasion, migration, and angiogenesis of HUVECs and CRC cells. CONCLUSION: Exosomal miR-522-3p from CAFs promoted the growth and metastasis of CRC through downregulating BMP5, which might provide new strategies for the treatment of CRC.

BMP5 ameliorates diabetic peripheral neuropathy by augmenting mitochondrial function and inhibiting apoptosis in Schwann cells.

Diabetic peripheral neuropathy is a common and serious complication of diabetes. Bone morphogenetic protein 5 (BMP5) is a multifunctional protein involved in the nervous system. Nevertheless, its effect on diabetic peripheral neuropathy remained uncharacterized. In this study, diabetic neuropathy in mice was induced by a single dose of 150 mg/kg streptozotocin (STZ) via intraperitoneal injection. Lentivirus expressing BMP5 (LV-BMP5) administration improved pain sensitivity, nerve conduction velocities and morphological alterations of the sciatic nerve of diabetic mice. Elevated BMP5 by LV-BMP5 suppressed cell apoptosis in the sciatic nerve, as evidenced by declined TUNEL-positive cells and down-regulated cleaved caspase-3 and cleaved caspase-9 levels. BMP5 enhanced mitochondrial membrane potential and ATP level. BMP5 also increased the phosphorylation of Smad1/5/9. Besides, the role of BMP5 in high glucose (HG)-stimulated Schwann cells was determined. Results of in vitro studies were in line with the in vivo findings. These experimental data seem to imply that BMP5 prevents the development of diabetic neuropathy via the maintenance of Smad1/5/9-mediated mitochondrial function.

Changes in the Transcriptome-Associated Co-Expression Profile of Embryonic External Ear Development After the BMP5 Gene Mutation.

This study aimed to perform an association analysis of the full transcriptome in Bmp5 short-ear mice during the development of the external ear in mouse embryos using advanced sequencing techniques. To understand the changes in gene regulation and expression of BMP5 gene mutations involved in the external ear embryonic development of mice, external ear tissues of mouse embryos developed to E15.5 and E17.5 were obtained using a BMP5 short-ear mouse model. The association analysis of the full transcriptome mainly involved the analysis of lncRNA and mRNA associations, the analysis of lncRNA and miRNA associations, the analysis of miRNA and mRNA associations, the analysis of circRNA and mRNA associations and circRNA, miRNA, and mRNA associations. The results showed that regulation of the full transcriptome is associated with external ear development in BMP5 short-ear mouse embryos, and some key regulatory changes in full transcriptome after BMP5 gene point mutation are different. This study will provide a new clue to investigate the mechanism underlying the regulation of mouse external ear development by the full transcriptome.

Bmp5 Mutation Alters mRNA Expression During External Ear Development.

To understand changes in gene regulation and mRNA expression in external ear development, we used a bone morphogenetic protein 5 (BMP5) short-ear mouse model. External ear tissues at E15.5 and E17.5 were collected, and mRNA expression profiles were analyzed. Upregulated and downregulated mRNA expression was identified using find_circ and CIRI2 software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed using the differentially expressed mRNAs. Alterations in related signal pathways were identified from the upregulated and downregulated mRNA transcripts. The results showed a correlation between the mRNA expression during external ear development in BMP5 short-ear mice, including key regulatory mRNA changes after point mutations of the Bmp5 gene. This study provides evidence for the mechanism underlying mRNA regulation during external ear development. Changes in mRNA expression profiles also provide clues for future studies regarding the regulatory mechanisms underlying external ear development.

Bmp5 Mutation Alters circRNA Expression During Embryonic External Ear Development.

ABSTRACT: Bmp5 mutation can lead to microtia in mice models; however, its underlying mechanism is unclear. We analyzed circular RNA (circRNA) expression changes and associated gene regulation during embryonic development of the mouse's external ear after a point mutation occurred naturally in the BMP5 gene. The outer ear tissues of BMP5 short-eared mouse model embryos at embryonic day (E) 15.5 and E17.5 were subjected to RNA sequencing. Changes in the circRNA expression profile were detected using find_circ and the CiRi2 software. Differentially expressed circRNAs were annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The circRNA expression profile differed between wild-type and mutant mouse embryos. At E15.5, differentially expressed RNAs were involved in the Hippo signaling pathway, whereas those at E17.5 were associated with stem cell pluripotency. Therefore, circRNA is involved in regulating embryonic external ear development, thus providing a basis for studying the biological aspect of its regulation.

Bmp5 Mutation Alters miRNA Expression During Embryonic External Ear Development.

ABSTRACT: To understand the changes in gene regulation and expression of MicroRNA (miRNA) involved in external mouseear embryonic development after point mutation of the Bmp5 gene, the outer ear tissues of developed E15.5 and E17.5 mouse embryos were obtained using a Bmp5 short ear mouse model, and the changes in miRNA expression profiles were detected. Changes in miRNA expression in the experimental and control groups were identified during Bmp5 short ear mouse embryo development at E15.5 and E17.5. GO and Kyoto Encyclopedia of Genes and Genomes functional annotations were performed on differentially expressed miRNAs. Multiple signal pathways related to miRNA expression were enhanced during the development of E15.5 and E17.5 embryos of Bmp5 short-ear mice. Based on the basic characteristics of miRNAs, this study aimed to determine the differential expression of miRNAs in Bmp5 short-ear mice during the development of external ear embryos using advanced sequencing techniques. The results showed differences in some key regulatory miRNA changes after point mutations in the Bmp5 gene. This study provides new insights into the mechanism by which miRNAs regulate the development of the external mouse ear. Changes in miRNA expression profiles can also provide clues for studying the biological regulatory mechanism of external ear embryonic development.

G9a Knockdown Suppresses Cancer Aggressiveness by Facilitating Smad Protein Phosphorylation through Increasing BMP5 Expression in Luminal A Type Breast Cancer.

Epigenetic abnormalities affect tumor progression, as well as gene expression and function. Among the diverse epigenetic modulators, the histone methyltransferase G9a has been focused on due to its role in accelerating tumorigenesis and metastasis. Although epigenetic dysregulation is closely related to tumor progression, reports regarding the relationship between G9a and its possible downstream factors regulating breast tumor growth are scarce. Therefore, we aimed to verify the role of G9a and its presumable downstream regulators during malignant progression of breast cancer. G9a-depleted MCF7 and T47D breast cancer cells exhibited suppressed motility, including migration and invasion, and an improved response to ionizing radiation. To identify the possible key factors underlying these effects, microarray analysis was performed, and a TGF-ß superfamily member, BMP5, was selected as a prominent target gene. It was found that BMP5 expression was markedly increased by G9a knockdown. Moreover, reduction in the migration/invasion ability of MCF7 and T47D breast cancer cells was induced by BMP5. Interestingly, a G9a-depletion-mediated increase in BMP5 expression induced the phosphorylation of Smad proteins, which are the intracellular signaling mediators of BMP5. Accordingly, we concluded that the observed antitumor effects may be based on the G9a-depletion-mediated increase in BMP5 expression and the consequent facilitation of Smad protein phosphorylation.

Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction.

Despite the high prevalence of ischemic heart diseases worldwide, no antibody-based treatment currently exists. Starting from the evidence that a specific isoform of the Bone Morphogenetic Protein 1 (BMP1.3) is particularly elevated in both patients and animal models of myocardial infarction, here we assess whether its inhibition by a specific monoclonal antibody reduces cardiac fibrosis. We find that this treatment reduces collagen deposition and cross-linking, paralleled by enhanced cardiomyocyte survival, both in vivo and in primary cultures of cardiac cells. Mechanistically, we show that the anti-BMP1.3 monoclonal antibody inhibits Transforming Growth Factor ß pathway, thus reducing myofibroblast activation and inducing cardioprotection through BMP5. Collectively, these data support the therapeutic use of anti-BMP1.3 antibodies to prevent cardiomyocyte apoptosis, reduce collagen deposition and preserve cardiac function after ischemia.

Whole-Genome Sequencing Identifies Two Novel Rare Mutations in BMP5 and BMP2 in Monozygotic Twins With Microtia.

ABSTRACT: Microtia is a rare congenital anomaly of the ear; it is regulated by both genetic and environmental factors. However, the mechanisms underlying its pathogenesis are unknown. In this study, the genomes of 2-year-old twin sisters with right microtia were sequenced using human genome-wide sequencing, an approach useful for identifying mutations in genes responsible for congenital microtia. The phenotypes of the twin sisters included congenital microtia on the right side, abnormal auricle shape in the right external ear, a peanut shape for the residual ear, and complete atresia of the right external auditory canal. In the twin sisters, we identified a previously unknown mutation in BMP5(exon4:c.833- 4C>G), as well as a new mutation (exon2:c.G332T:p.S111I) in BMP2, both of which were confirmed using polymerase chain reaction-based amplification of the corresponding genome regions, followed by first-generation sequencing. The exon4:c.833-4C>G mutation in human BMP5 may be the main cause of microtia in the twin sisters. A pathogenic mutation in human BMP2 (exon2:c.G332T:p.S111I) may be responsible for the facial deformity in the twin sisters. Thus, our study demonstrates the potential of genome-wide sequencing for identifying novel mutations associated with microtia on the whole-genome scale and extends the mutation spectrum of BMP5. Additionally, our data suggest that BMP2 is another pathogenic gene associated with microtia.

Upregulated bone morphogenetic protein 5 enhances proliferation and epithelial-mesenchymal transition process in benign prostatic hyperplasia via BMP/Smad signaling pathway.

BACKGROUND: Benign prostatic hyperplasia (BPH) is one of the most common illnesses in aging men. Recent studies found that bone morphogenetic protein 5 (BMP5) is upregulated in BPH tissues, however, the role of BMP5 in the development of BPH has not been examined. The current study aims to elucidate the potential roles of BMP5 and related signaling pathways in BPH. METHODS: Human prostate cell lines (BPH-1, WPMY-1) and human/rat hyperplastic prostate tissues were utilized. Western blot, quantitative real-time polymerase chain reaction, immunofluorescent staining, and immunohistochemical staining were performed. BMP5-silenced and -overexpressed cell models were generated and then cell cycle progression, apoptosis, and proliferation were determined. The epithelial-mesenchymal transition (EMT) was also quantitated. And rescue experiments by BMP/Smad signaling pathway agonist or antagonist were accomplished. Moreover, BPH-related tissue microarray analysis was performed and associations between clinical parameters and expression of BMP5 were analyzed. RESULTS: Our study demonstrated that BMP5 was upregulated in human and rat hyperplastic tissues and localized both in the epithelial and stromal compartments of the prostate tissues. E-cadherin was downregulated in hyperplastic tissues, while N-cadherin and vimentin were upregulated. Overexpression of BMP5 enhanced cell proliferation and the EMT process via phosphorylation of Smad1/5/8, while knockdown of BMP5 induced cell cycle arrest at G0/G1 phase and blocked the EMT process. Moreover, a BMP/Smad signaling pathway agonist and antagonist reversed the effects of BMP5 silencing and overexpression, respectively. In addition, BMP5 expression positively correlated with prostate volume and total prostate-specific antigen. CONCLUSION: Our novel data suggest that BMP5 modulated cell proliferation and the EMT process through the BMP/Smad signaling pathway which could contribute to the development of BPH. However, further studies are required to determine the exact mechanism. Our study also indicated that BMP/Smad signaling may be rediscovered as a promising new therapeutic target for the treatment of BPH.

Identifying molecular pathways and candidate genes associated with knob traits by transcriptome analysis in the goose (Anser cygnoides).

Anser cygnoides has a spherical crest on the beak roof, which is described as knob. However, the mechanisms affecting knob morphology are unclear. Here, we investigated the phenotypic characteristics and molecular basis of knob-size differences in Yangzhou geese. Anatomically, the knob was identified as frontal hump in the frontal area of the skull, rather than hump of upper beak. Although the frontal hump length, and height varied greatly in geese with different knob phenotypes, little was changed in the width. Histologically, knob skin in large-size knobs geese have a greater length in the stratum corneum, stratum spinosum, and stratum reticular than that in small-size knobs geese. Moveover, the 415 differentially expressed genes were found between the large knobs and small ones through transcriptome profiling. In addition, GO enrichment and KEGG pathway analysis revealed 455 significant GO terms and 210 KEGG pathways were enriched, respectively. Among these, TGF-ß signaling and thyroid hormone synthesis-signaling pathways were identified to determine knob-size phenotype. Furthermore, BMP5, DCN, TSHR and ADCY3 were recognized to involve in the growth and development of knob. Our data provide comprehensive molecular determinants of knob size phenotype, which can potentially promote the genetic improvement of goose knobs.

BMP5 silencing inhibits chondrocyte senescence and apoptosis as well as osteoarthritis progression in mice.

In this study, we using the in vivo destabilization of the medial meniscus (DMM) mouse model to investigate the role of bone morphogenetic protein 5 (BMP5) in osteoarthritis (OA) progression mediated via chondrocyte senescence and apoptosis. BMP5 expression was significantly higher in knee articular cartilage tissues of OA patients and DMM model mice than the corresponding controls. The Osteoarthritis Research Society International scores based on histological staining of knee articular cartilage sections were lower in DMM mice where BMP5 was knocked down in chondrocytes than the corresponding controls 4 weeks after DMM surgery. DMM mice with BMP5-deficient chondrocytes showed reduced levels of matrix-degrading enzymes such as MMP13 and ADAMTS5 as well as reduced cartilage destruction. BMP5 knockdown also decreased chondrocyte apoptosis and senescence by suppressing the activation of p38 and ERK MAP kinases. These findings demonstrate that BMP5 silencing inhibits chondrocyte senescence and apoptosis as well as OA progression by downregulating activity in the p38/ERK signaling pathway.

Detection of circulating BMP5 as a risk factor for Barrett's esophagus.

Barrett's esophagus (BE) predisposes for the malignant condition of esophageal adenocarcinoma (EAC). Since BE patients have few or no symptoms, most of these patients are not identified and not included in surveillance programs. These BE patients are at risk of developing advanced-stage EAC. At present, non-invasive tests to identify BE patients from the general population are lacking. We and others showed that Bone Morphogenetic Protein 4 (BMP4), and other BMPs are upregulated in BE. We aimed to determine if circulating BMPs can be identified and used as blood biomarkers to identify BE patients at high risk in the general population. In this study, we could detect the different BMPs in the blood of 112 BE patients and 134 age- and sex-matched controls. Concentration levels of BMP2, BMP4, and BMP5 were elevated in BE patients, with BMP2 and BMP5 significantly increased. BMP5 remained significant after multivariate analysis and was associated with an increased risk for BE with an OR of 1.49 (p value 0.01). Per log (pg/mL) of BMP5, the odds of having BE increased by 50%. Future optimization and validation studies might be needed to prove its utility as a non-invasive method for the detection of BE in high-risk populations and screening programs.

Regulation of stem/progenitor cell maintenance by BMP5 in prostate homeostasis and cancer initiation.

Tissue homeostasis relies on the fine regulation between stem and progenitor cell maintenance and lineage commitment. In the adult prostate, stem cells have been identified in both basal and luminal cell compartments. However, basal stem/progenitor cell homeostasis is still poorly understood. We show that basal stem/progenitor cell maintenance is regulated by a balance between BMP5 self-renewal signal and GATA3 dampening activity. Deleting Gata3 enhances adult prostate stem/progenitor cells self-renewal capacity in both organoid and allograft assays. This phenotype results from a local increase in BMP5 activity in basal cells as shown by the impaired self-renewal capacity of Bmp5-deficient stem/progenitor cells. Strikingly, Bmp5 gene inactivation or BMP signaling inhibition with a small molecule inhibitor are also sufficient to delay prostate and skin cancer initiation of Pten-deficient mice. Together, these results establish BMP5 as a key regulator of basal prostate stem cell homeostasis and identifies a potential therapeutic approach against Pten-deficient cancers.

Genomic analysis of benign prostatic hyperplasia implicates cellular re-landscaping in disease pathogenesis.

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.

Direct reprogramming of epidermal cells toward sweat gland-like cells by defined factors.

Several studies have reported inducing adult cells into sweat gland-like cells; however, slow transition and low efficiency limit the potential for cell-based treatment. Here, we show that overexpression of the transcription factor FoxC1 was sufficient to reprogram epidermal cells to induced functional sweat gland-like cells (iSGCs). The iSGCs expressing secreting-related genes, had a global gene expression profile between fetal SGCs (P5) and adult SGCs (P28). Moreover, iSGCs transplanted into the burn mice model facilitated wound repair and sweat gland regeneration. We further demonstrated that the Foxc1 upregulated BMP5 transcription and BMP5 is responsible for the cell-type transition. Collectively, this study shows that lineage reprogramming of epidermal cells into iSGCs provides an excellent cell source and a promising regenerative strategy for anhidrosis and hypohidrosis.

Alteration of tumor suppressor BMP5 in sporadic colorectal cancer: a genomic and transcriptomic profiling based study.

BACKGROUND: Although the genetic spectrum of human colorectal cancer (CRC) is mainly characterized by APC, KRAS and TP53 mutations, driver genes in tumor initiation have not been conclusively demonstrated. In this study, we aimed to identify novel markers for CRC. METHODS: We performed exome analysis of sporadic colorectal cancer (sCRC) coding regions to screen loss of function (LoF) mutation genes, and carried out systems-level approaches to confirm top rank gene in this study. RESULTS: We identified loss of BMP5 is an early event in CRC. Deep sequencing identified BMP5 was mutated in 7.7% (8/104) of sCRC samples, with 37.5% truncating mutation frequency. Notably, BMP5 negative expression and its prognostic value is uniquely significant in sCRC but not in other tumor types. Furthermore, BMP5 expression was positively correlated with E-cadherin in CRC patients and its dysregulation play a vital role in epithelial-mesenchymal transition (EMT), thus triggering tumor initiation and development. RNA sequencing identified, independent of BMP/Smads pathway, BMP5 signaled though Jak-Stat pathways to inhibit the activation of oncogene EPSTI1. CONCLUSIONS: Our result support a novel concept that the importance of BMP5 in sCRC. The tumor suppressor role of BMP5 highlights its crucial role in CRC initiation and development.

Bone marrow-derived epithelial cells and hair follicle stem cells contribute to development of chronic cutaneous neoplasms.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.

A Pan-Cancer Analysis Reveals High-Frequency Genetic Alterations in Mediators of Signaling by the TGF-ß Superfamily.

We present an integromic analysis of gene alterations that modulate transforming growth factor ß (TGF-ß)-Smad-mediated signaling in 9,125 tumor samples across 33 cancer types in The Cancer Genome Atlas (TCGA). Focusing on genes that encode mediators and regulators of TGF-ß signaling, we found at least one genomic alteration (mutation, homozygous deletion, or amplification) in 39% of samples, with highest frequencies in gastrointestinal cancers. We identified mutation hotspots in genes that encode TGF-ß ligands (BMP5), receptors (TGFBR2, AVCR2A, and BMPR2), and Smads (SMAD2 and SMAD4). Alterations in the TGF-ß superfamily correlated positively with expression of metastasis-associated genes and with decreased survival. Correlation analyses showed the contributions of mutation, amplification, deletion, DNA methylation, and miRNA expression to transcriptional activity of TGF-ß signaling in each cancer type. This study provides a broad molecular perspective relevant for future functional and therapeutic studies of the diverse cancer pathways mediated by the TGF-ß superfamily.