Role of Hedgehog Signaling Pathways in Multipotent Mesenchymal Stem Cells Differentiation.

Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much attention in the field of regenerative medicine and have a promising clinical application. MSCs originate from the mesoderm and can differentiate not only into osteoblasts, cartilage, adipocytes, and muscle cells but also into ectodermal and endodermal cell lineages across embryonic layers. To design cell therapy for replacement of damaged tissues, it is essential to understand the signaling pathways, which have a major impact on MSC differentiation, as this will help to integrate the signaling inputs to initiate a specific lineage. Hedgehog (Hh) signaling plays a vital role in the development of various tissues and organs in the embryo. As a morphogen, Hh not only regulates the survival and proliferation of tissue progenitor and stem populations but also is a critical moderator of MSC differentiation, involving tri-lineage and across embryonic layer differentiation of MSCs. This review summarizes the role of Hh signaling pathway in the differentiation of MSCs to mesodermal, endodermal, and ectodermal cells.

A Pilot Immunohistochemical Study Identifies Hedgehog Pathway Expression in Sinonasal Adenocarcinoma.

Tumors of the head and neck, more specifically the squamous cell carcinoma, often show upregulation of the Hedgehog signaling pathway. However, almost nothing is known about its role in the sinonasal adenocarcinoma, either in intestinal or non-intestinal subtypes. In this work, we have analyzed immunohistochemical staining of six Hedgehog pathway proteins, sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched1 (PTCH1), Gli family zinc finger 1 (GLI1), Gli family zinc finger 2 (GLI2), and Gli family zinc finger 3 (GLI3), on 21 samples of sinonasal adenocarcinoma and compared them with six colon adenocarcinoma and three salivary gland tumors, as well as with matching healthy tissue, where available. We have detected GLI2 and PTCH1 in the majority of samples and also GLI1 in a subset of samples, while GLI3 and the ligands SHH and IHH were generally not detected. PTCH1 pattern of staining shows an interesting pattern, where healthy samples are mostly positive in the stromal compartment, while the signal shifts to the tumor compartment in tumors. This, taken together with a stronger signal of GLI2 in tumors compared to non-tumor tissues, suggests that the Hedgehog pathway is indeed activated in sinonasal adenocarcinoma. As Hedgehog pathway inhibitors are being tested in combination with other therapies for head and neck squamous cell carcinoma, this could provide a therapeutic option for patients with sinonasal adenocarcinoma as well.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

RSG1 is required for cilia-dependent neural tube closure.

Cilia play a key role in the regulation of signaling pathways required for embryonic development, including the proper formation of the neural tube, the precursor to the brain and spinal cord. Forward genetic screens were used to generate mouse lines that display neural tube defects (NTD) and secondary phenotypes useful in interrogating function. We describe here the L3P mutant line that displays phenotypes of disrupted Sonic hedgehog signaling and affects the initiation of cilia formation. A point mutation was mapped in the L3P line to the gene Rsg1, which encodes a GTPase-like protein. The mutation lies within the GTP-binding pocket and disrupts the highly conserved G1 domain. The mutant protein and other centrosomal and IFT proteins still localize appropriately to the basal body of cilia, suggesting that RSG1 GTPase activity is not required for basal body maturation but is needed for a downstream step in axonemal elongation.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

BACKGROUND: Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). METHODS AND RESULTS: HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-ß. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). CONCLUSIONS: YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

ERß Regulation of Indian Hedgehog Expression in the First Wave of Ovarian Follicles.

Increased activation of ovarian primordial follicles in Erß knockout (ErßKO) rats becomes evident as early as postnatal day 8.5. To identify the ERß-regulated genes that may control ovarian primordial follicle activation, we analyzed the transcriptome profiles of ErßKO rat ovaries collected on postnatal days 4.5, 6.5, and 8.5. Compared to wildtype ovaries, ErßKO ovaries displayed dramatic downregulation of Indian hedgehog (Ihh) expression. IHH-regulated genes, including Hhip, Gli1, and Ptch1, were also downregulated in ErßKO ovaries. This was associated with a downregulation of steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1. The expression of Ihh remained very low in ErßKO ovaries despite the high levels of Gdf9 and Bmp15, which are known upregulators of Ihh expression in the granulosa cells of activated ovarian follicles. Strikingly, the downregulation of the Ihh gene in ErßKO ovaries began to disappear on postnatal day 16.5 and recovered on postnatal day 21.5. In rat ovaries, the first wave of primordial follicles is rapidly activated after their formation, whereas the second wave of primordial follicles remains dormant in the ovarian cortex and slowly starts activating after postnatal day 12.5. We localized the expression of Ihh mRNA in postnatal day 8.5 wildtype rat ovaries but not in the age-matched ErßKO ovaries. In postnatal day 21.5 ErßKO rat ovaries, we detected Ihh mRNA mainly in the activated follicles in the ovaries' peripheral regions. Our findings indicate that the expression of Ihh in the granulosa cells of the activated first wave of ovarian follicles depends on ERß.

Human umbilical cord mesenchymal stem cell-derived exosomes ameliorate renal fibrosis in diabetic nephropathy by targeting Hedgehog/SMO signaling.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease globally. Currently, there are no effective drugs for the treatment of DN. Although several studies have reported the therapeutic potential of mesenchymal stem cells, the underlying mechanisms remain largely unknown. Here, we report that both human umbilical cord MSCs (UC-MSCs) and UC-MSC-derived exosomes (UC-MSC-exo) attenuate kidney damage, and inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis in streptozotocin-induced DN rats. Strikingly, the Hedgehog receptor, smoothened (SMO), was significantly upregulated in the kidney tissues of DN patients and rats, and positively correlated with EMT and renal fibrosis. UC-MSC and UC-MSC-exo treatment resulted in decrease of SMO expression. In vitro co-culture experiments revealed that UC-MSC-exo reduced EMT of tubular epithelial cells through inhibiting Hedgehog/SMO pathway. Collectively, UC-MSCs inhibit EMT and renal fibrosis by delivering exosomes and targeting Hedgehog/SMO signaling, suggesting that UC-MSCs and their exosomes are novel anti-fibrotic therapeutics for treating DN.

Dual and opposing roles for the kinesin-2 motor, KIF17, in Hedgehog-dependent cerebellar development.

While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion partially phenocopies Kif17 germline mutants. Unexpectedly, CGNP-specific Kif17 deletion results in the opposite phenotype-increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together, these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPs.

Epigenetic regulation by KDM5A mediates the effects of prenatal PM2.5 exposure on hippocampal development and synaptic integrity through the Shh signaling pathway.

Prenatal environmental exposure could be an essential health risk factor associated with neurodevelopmental disorders in offspring. However, the exact mechanisms underlying the impact of prenatal PM2.5 exposure on offspring cognition remain unclear. In our recent study using a PM2.5 exposed pregnant mouse model, we observed significant synaptic dysfunction in the hippocampi of the offspring. Concurrently, the epigenetic regulator of KDM5A and the Shh signaling pathway exhibited decreased activities. Significantly, changes in hippocampal KDM5A and Shh levels directly correlated with PM2.5 exposure intensity. Subsequent experiments revealed a marked reduction in the expression of Shh signaling and related synaptic proteins when KDM5A was silenced in cells. Notably, the effects of KDM5A deficiency were reversed significantly with the supplementation of a Shh activator. Furthermore, our findings indicate that Shh activation significantly attenuates PM2.5-induced synaptic impairments in hippocampal neurons. We further demonstrated that EGR1, a transcriptional inhibitor, plays a direct role in KDM5A's regulation of the Shh pathway under conditions of PM2.5 exposure. Our results suggest that the KDM5A's inhibitory regulation on the Shh pathway through the EGR1 gene is a crucial epigenetic mechanism underlying the synaptic dysfunction in hippocampal neurons caused by maternal PM2.5 exposure. This emphasizes the role of epigenetic regulations in neurodevelopmental disorders caused by environmental factors.

The importance of caregivers for patients with advanced basal cell carcinoma treated with hedgehog-pathway inhibitors: an observational prospective study.

Oral targeted therapy with hedgehog pathway inhibitors has revolutionized the standard of care for patients with advanced basal cell carcinoma (BCC). These patients are frail and elderly, have various comorbidities, and receive pharmacological polytherapy. Moreover, adverse events may have a significant impact on therapeutic adherence, which must be managed by the clinician. We evaluated the impact of caregivers on the treatment of patients with advanced BCC in terms of continuation of therapy over time. All patients included in this observational prospective study had histologically confirmed metastatic or locally advanced BCC (LaBCC) and were treated with hedgehog pathway inhibitors from January 2016 to December 2021 at the Department of Dermatology at the University of Florence, Italy. The collected patient data included: age, sex, BCC site and area of spread; number of cycles, dose, duration and tolerability of therapy; marital status (single, divorced, married/living with a partner, widow/widower); and information such as living with someone, and the presence of any caregivers. Of the 34 patients included, 33 had LaBCC and one metastatic BCC. There were 11 females (32.4%) and 23 males (67.6%). Patients who were married or living with a caregiver -tolerated therapy better than single patients who lived alone. Indeed, patients with married/live-in caregivers and/or those with an adequate caregiver experienced greater therapeutic adherence and tolerance of adverse events. Given the greater therapeutic adherence of patients with live-in caregivers as partners, it is essential to consider patients' marital status. It is advisable to involve the caregiver early on, and there should be a training discussion on the various possible adverse events and the best way to mitigate them. Therapeutic success is linked not only to patients being informed but also to training of caregivers.

Hepatic progenitor cell-originated ductular reaction facilitates liver fibrosis through activation of hedgehog signaling.

Background: It is poorly understood what cellular types participate in ductular reaction (DR) and whether DR facilitates recovery from injury or accelerates hepatic fibrosis. The aim of this study is to gain insights into the role of hepatic progenitor cell (HPC)-originated DR during fibrotic progression. Methods: DR in liver specimens of PBC, chronic HBV infection (CHB) or NAFLD, and four rodent fibrotic models by different pathogenic processes was evaluated. Gli1 expression was inhibited in rodent models or cell culture and organoid models by AAV-shGli1 or treating with GANT61. Results: Severity of liver fibrosis was positively correlated with DR extent in patients with PBC, CHB or NAFLD. HPCs were activated, expanded, differentiated into reactive cholangiocytes and constituted "HPC-originated DR", accompanying with exacerbated fibrosis in rodent models of HPC activation & proliferation (CCl4/2-AAF-treated), Μdr2-/- spontaneous PSC, BDL-cholestatic fibrosis or WD-fed/CCl4-treated NASH-fibrosis. Gli1 expression was significantly increased in enriched pathways in vivo and in vitro. Enhanced Gli1 expression was identified in KRT19+-reactive cholangiocytes. Suppressing Gli1 expression by administration of AAV-shGli1 or GANT61 ameliorated HPC-originated DR and fibrotic extent. KRT19 expression was reduced after GANT61 treatment in sodium butyrate-stimulated WB-F344 cells or organoids or in cells transduced with Gli1 knockdown lentiviral vectors. In contrast, KRT19 expression was elevated after transducing Gli1 overexpression lentiviral vectors in these cells. Conclusions: During various modes of chronic injury, Gli1 acted as an important mediator of HPC activation, expansion, differentiation into reactive cholangiocytes that formed DR, and subsequently provoked hepatic fibrogenesis.

CBC-1 as a Cynanbungeigenin C derivative inhibits the growth of colorectal cancer through targeting Hedgehog pathway component GLI 1.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers that results in death in worldwide. The Hedgehog (HH) signalling pathway regulates the initiation and progression of CRC. Inhibiting the HH pathway has been presented as a potential treatment strategy in recent years. Cynanbungeigenin C (CBC) is a new type of C21 steroid that has been previously reported for the treatment of medulloblastoma. However, its further investigation was limited by its poor water solubility. In this study, six new CBC derivatives were synthesized through the structural modification of CBC, and four of them showed better water solubility than CBC. Moreover, their antiproliferative activities on CRC were evaluated. It was found that CBC-1 presented the best inhibitory effect on three types of CRC cell lines, and this effect was superior to that of CBC. Mechanistically, CBC-1 inhibited the proliferation of CRC cells through regulation of mRNA and proteins of the HH pathway according to qRT-PCR and Western blotting analysis. Furthermore, Cellular Thermal Shift Assay results indicated that CBC-1 regulated this signalling pathway by targeting glioma­associated oncogene (GLI 1).In addition, cell apoptosis was induced increasingly by transfection with GLI 1 siRNA or treatment with CBC-1 to downregulate GLI 1. Last, the in vivo results demonstrated that CBC-1 significantly reduced tumour size and downregulated GLI 1 in CRC. Therefore, this study suggests that CBC-1, a new GLI 1 inhibitor derived from natural products, may be developed as a potential antitumour candidate for CRC treatment.

LOXL1-AS1 contributes to metastasis in sonic-hedgehog medulloblastoma by promoting cancer stem-like phenotypes.

BACKGROUND: Medulloblastomas (MBs) are one of the most common malignant brain tumor types in children. MB prognosis, despite improvement in recent years, still depends on clinical and biological risk factors. Metastasis is the leading cause of MB-related deaths, which highlights an unmet need for risk stratification and targeted therapy to improve clinical outcomes. Among the four molecular subgroups, sonic-hedgehog (SHH)-MB harbors clinical and genetic heterogeneity with a subset of high-risk cases. Recently, long non-coding (lnc)RNAs were implied to contribute to cancer malignant progression, but their role in MB remains unclear. This study aimed to identify pro-malignant lncRNAs that have prognostic and therapeutic significance in SHH-MB. METHODS: The Daoy SHH-MB cell line was engineered for ectopic expression of MYCN, a genetic signature of SHH-MB. MYCN-associated lncRNA genes were identified using RNA-sequencing data and were validated in SHH-MB cell lines, MB tissue samples, and patient cohort datasets. SHH-MB cells with genetic manipulation of the candidate lncRNA were evaluated for metastatic phenotypes in vitro, including cell migration, invasion, sphere formation, and expressions of stemness markers. An orthotopic xenograft mouse model was used to evaluate metastasis occurrence and survival. Finally, bioinformatic screening and in vitro assays were performed to explore downstream mechanisms. RESULTS: Elevated lncRNA LOXL1-AS1 expression was identified in MYCN-expressing Daoy cells and MYCN-amplified SHH-MB tumors, and was significantly associated with lower survival in SHH-MB patients. Functionally, LOXL1-AS1 promoted SHH-MB cell migration and cancer stemness in vitro. In mice, MYCN-expressing Daoy cells exhibited a high metastatic rate and adverse effects on survival, both of which were suppressed under LOLX1-AS1 perturbation. Integrative bioinformatic analyses revealed associations of LOXL1-AS1 with processes of cancer stemness, cell differentiation, and the epithelial-mesenchymal transition. LOXL1-AS1 positively regulated the expression of transforming growth factor (TGF)-ß2. Knockdown of TGF-ß2 in SHH-MB cells significantly abrogated their LOXL1-AS1-mediated prometastatic functions. CONCLUSIONS: This study proved the functional significance of LOXL1-AS1 in SHH-MB metastasis by its promotion of TGF-ß2-mediated cancer stem-like phenotypes, providing both prognostic and therapeutic potentials for targeting SHH-MB metastasis.

Hedgehog pathway inhibitors for locally advanced and metastatic basal cell carcinoma: A real-world single-center retrospective review.

Basal cell carcinoma (BCC) is highly curable by surgical excision or radiation. In rare cases, BCC can be locally destructive or difficult to surgically remove. Hedgehog inhibition (HHI) with vismodegib or sonidegib induces a 50-60% response rate. Long-term toxicity includes muscle spasms and weight loss leading to dose decreases. This retrospective chart review also investigates the impact of CoQ10 and calcium supplementation in patients treated with HHI drugs at a single academic medical center from 2012 to 2022. We reviewed the charts of adult patients diagnosed with locally advanced or metastatic BCC treated with vismodegib or sonidegib primarily for progression-free survival (PFS). Secondary objectives included overall survival, BCC-specific survival, time to and reasons for discontinuation, overall response rate, safety and tolerability, use of CoQ10 and calcium supplements, and insurance coverage. Of 55 patients assessable for outcome, 34 (61.8%) had an overall clinical benefit, with 25 (45.4%) having a complete response and 9 (16.3%) a partial response. Stable disease was seen in 14 (25.4%) and 7 (12.7%) progressed. Of the 34 patients who responded to treatment, 9 recurred. Patients who were rechallenged with HHI could respond again. The median overall BCC-specific survival rate at 5 years is 89%. Dose reductions or discontinuations for vismodegib and sonidegib occurred in 59% versus 24% of cases, or 30% versus 9% of cases, respectively. With CoQ10 and calcium supplementation, only 17% required a dose reduction versus 42% without. HHI is highly effective for treating advanced BCC but may require dosing decreases. Sonidegib was better tolerated than vismodegib. CoQ10 and calcium supplementation can effectively prevent muscle spasms.

Pilose antler extract promotes hair growth in androgenic alopecia mice by promoting the initial anagen phase.

Androgenetic alopecia (AGA) is a prevalent disease in worldwide, local application or oral are often used to treat AGA, however, effective treatments for AGA are currently limited. In this work, we observed the promoting the initial anagen phase effect of pilose antler extract (PAE) on hair regeneration in AGA mice. We found that PAE accelerated hair growth and increased the degree of skin blackness by non-invasive in vivo methods including camera, optical coherence tomography and dermoscopy. Meanwhile, HE staining of sagittal and coronal skin sections revealed that PAE augmented the quantity and length of hair follicles, while also enhancing skin thickness and hair papilla diameter. Furthermore, PAE facilitated the shift of the growth cycle from the telogen to the anagen phase and expedited the proliferation of hair follicle stem cells and matrix cells in mice with AGA. This acceleration enabled the hair follicles to enter the growth phase at an earlier stage. PAE upregulated the expression of the sonic hedgehog (SHH), smoothened receptor, glioma-associated hemolog1 (GLI1), and downregulated the expression of bone morphogenetic protein 4 (BMP4), recombinant mothers against decapentaplegic homolog (Smad) 1 and 5 phosphorylation. This evidence suggests that PAE fosters hair growth and facilitates the transition of the growth cycle from the telogen to the anagen phase in AGA mice. This effect is achieved by enhancing the proliferation of follicle stem cells and matrix cells through the activation of the SHH/GLI pathway and suppression of the BMP/Smad pathway.

SHH regulates penile morphology and smooth muscle through a mechanism involving BMP4 and GREM1.

BACKGROUND: The cavernous nerve (CN) is frequently damaged in prostatectomy and diabetic patients with erectile dysfunction (ED), initiating changes in penile morphology including an acute and intense phase of apoptosis in penile smooth muscle and increased collagen, which alter penile architecture and make corpora cavernosa smooth muscle less able to relax in response to neurotransmitters, resulting in ED. AIM: Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and SHH treatment suppresses penile remodeling after CN injury through an unknown mechanism; we examine if part of the mechanism of how SHH preserves smooth muscle after CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1). METHODS: Primary cultures of smooth muscle cells were established from prostatectomy, diabetic, hypertension and Peyronie's (control) (N = 18) patients. Cultures were characterized by ACTA2, CD31, P4HB, and nNOS immunohistochemical analysis. Patient smooth muscle cell growth was quantified in response to BMP4 and GREM1 treatment. Adult Sprague Dawley rats underwent 1 of 3 surgeries: (1) uninjured or CN-injured rats were treated with BMP4, GREM1, or mouse serum albumin (control) proteins via Affi-Gel beads (N = 16) or peptide amphiphile (PA) (N = 26) for 3 and 14 days, and trichrome stain was performed; (2) rats underwent sham (N = 3), CN injury (N = 9), or CN injury and SHH PA treatment for 1, 2, and 4 days (N = 9). OUTCOMES: Western analysis for BMP4 and GREM1 was performed; (3) rats were treated with 5E1 SHH inhibitor (N = 6) or IgG (control; N = 6) for 2 and 4 days, and BMP4 and GREM1 localization was examined. Statistics were performed by analysis of variance with Scheffé's post hoc test. RESULTS: BMP4 increased patient smooth muscle cell growth, and GREM1 decreased growth. In rats, BMP4 treatment via Affi-Gel beads and PA increased smooth muscle at 3 and 14 days of treatment. GREM1 treatment caused increased collagen and smooth muscle at 3 days, which switched to primarily collagen at 14 days. CN injury increased BMP4 and GREM1, while SHH PA altered Western band size, suggesting alternative cleavage and range of BMP4 and GREM1 signaling. SHH inhibition in rats increased BMP4 and GREM1 in fibroblasts. CLINICAL IMPLICATIONS: Understanding how SHH PA preserves and regenerates penile morphology after CN injury will aid development of ED therapies. STRENGTHS AND LIMITATIONS: SHH treatment alters BMP4 and GREM1 localization and range of signaling, which can affect penile morphology. CONCLUSION: Part of the mechanism of how SHH regulates corpora cavernosa smooth muscle involves BMP4 and GREM1.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

BACKGROUND: Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. AIM: We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. METHODS: Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N = 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7 days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14 days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4 days; or (4) CN crush with mouse serum albumin or SHH for 9 days. OUTCOMES: Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. RESULTS: BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2 days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. CLINICAL IMPLICATIONS: A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. STRENGTHS AND LIMITATIONS: The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. CONCLUSION: BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

The comprehensive landscape of prognosis, immunity, and function of the GLI family by pan-cancer and single-cell analysis.

The Hedgehog (Hh) signaling pathway has been implicated in the pathogenesis of various cancers. However, the roles of the downstream GLI family (GLI1, GLI2, and GLI3) in tumorigenesis remain elusive. This study aimed to unravel the genetic alterations of GLI1/2/3 in cancer and their association with the immune microenvironment and related signaling pathways. Firstly, we evaluated the expression profiles of GLI1/2/3 in different cancer types, analyzed their prognostic and predictive values, and assessed their correlation with tumor-infiltrating immune cells. Secondly, we explored the relationships between GLI1/2/3 and genetic mutations, epigenetic modifications, and clinically relevant drugs. Finally, we performed enrichment analysis to decipher the underlying mechanisms of GLI1/2/3 in cancer initiation and progression. Our results revealed that the expression levels of GLI1/2/3 were positively correlated in most cancer tissues, suggesting a cooperative role of these factors in tumorigenesis. We also identified tissue-specific expression patterns of GLI1/2/3, which may reflect the distinct functions of these factors in different cell types. Furthermore, GLI1/2/3 expression displayed significant associations with poor prognosis in several cancers, indicating their potential as prognostic biomarkers and therapeutic targets. Importantly, we found that GLI1/2/3 modulated the immune microenvironment by regulating the recruitment, activation, and polarization of cancer-associated fibroblasts, endothelial cells, and macrophages. Additionally, functional enrichment analyses indicated that GLI1/2/3 are involved in the regulation of epithelial-mesenchymal transition (EMT). Together, our findings shed new light on the roles of GLI1/2/3 in tumorigenesis and provide a potential basis for the development of novel therapeutic strategies targeting GLI-mediated signaling pathways in cancer.

Childhood cancer mutagenesis caused by transposase-derived PGBD5.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

Identification of an early survival prognostic gene signature for localized osteosarcoma patients.

Osteosarcoma is the most prevalent bone tumor in pediatric patients. Neoadjuvant chemotherapy has improved osteosarcoma patient survival, however the 5-year survival rate for localized osteosarcoma is 75% with a 30-50% recurrence rate. We, therefore, sought to identify a prognostic gene signature which could predict poor prognosis in localized osteosarcoma patients. Using the TARGET osteosarcoma transcriptomic dataset, we identified a 13-hub gene signature associated with overall survival and time to death of localized osteosarcoma patients, with the high-risk group showing a 22% and the low-risk group showing 100% overall survival. Furthermore, network analysis identified five modules of co-expressed genes that significantly correlated with survival, and identified 65 pathways enriched across 3 modules, including Hedgehog signaling, which includes 2 of the 13 genes, IHH and GLI1. Subsequently, we demonstrated that GLI antagonists inhibited growth of a recurrent localized PDX-derived cell line with elevated IHH and GLI1 expression, but not a non-relapsed cell line with low pathway activation. Finally, we show that our signature outperforms previously reported signatures in predicting poor prognosis and death within 3 years in patients with localized osteosarcoma.